Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.     

Sphingomonas herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide degrader

Sphingomonas herbicidovorans MH was isolated from a dichlorprop-degrading soil column. It is able to grow on phenoxyalkanoic acid herbicides, such as mecoprop, dichlorprop, 2,4-D, MCPA, and 2,4-DB. Strain MH utilizes both enantiomers of the chiral herbicides mecoprop and dichlorprop as sole carbon and energy sources. Enantiomer-specific uptake systems are responsible for transporting the acidic substrates across the cell membrane. Catabolism is initiated by two enantiomer-specific α-ketoglutarate-dependent dioxygenases that catalyze the cleavage of the ether bond of the respective enantiomer to yield the corresponding phenol and pyruvate. Therefore selective degradation of the enantiomers of mecoprop and dichlorprop by strain MH is not only due to enantioselective catabolism but also to enantioselective transport. Received 07 May 1999/ Accepted in revised form 11 August 1999     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.     

Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(RS)-2-(2,4-Dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH

Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (K_t) of 108, 93, and 117 μM and maximal velocities (V_max) of 19, 10, and 21 nmol min⁻¹ mg of protein⁻¹ for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

Chlorophenol degradation coupled to sulfate reduction.

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Chlorophenol degradation coupled to sulfate reduction

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Separation of two dichlorprop/α-ketoglutarate dioxygenases with enantiospecific properties from Comamonas acidovorans MC1

Comamonas acidovorans MC1, which is capable of degrading the chiral phenoxypropionate herbicides 2-(2,4-dichlorophenoxy)propionate [dichlorprop, (RS)-2,4-DP] and 2-(4-chloro-2-methylphenoxy)propionate [mecoprop, (RS)-MCPP] and of degrading the phenoxyacetate herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), was investigated with respect to the enzymatic basis of this broad substrate specificity. The initial steps of the degradation pathway of (RS)-2,4-DP and 2,4-D were studied. By applying either ion exchange chromatography or hydrophobic interaction chromatography it was possible to separate two enzyme fractions with etherolytic activity, which exhibited pronounced substrate specificity. One enzyme fraction was highly specific for the degradation of the R-enantiomer of 2,4-DP and did not essentially attack the S-configuration. The other enzyme fraction showed pronounced activity toward the cleavage of the S-enantiomer and additionally utilized 2,4-D with almost equal velocity; (R)-2,4-DP was even cleaved at a low rate by this enzyme. These results confirm the existence of phenoxyalkanoatedegrading enzymes with enantiospecific properties in strain MC1.     

Effects of sorption on biological degradation rates of (2,4-dichlorophenoxy) acetic acid in soils.

Three mathematical models were proposed to describe the effects of sorption of both bacteria and the herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) on the biological degradation rates of 2,4-D in soils. Model 1 assumed that sorbed 2,4-D is not degraded, that only bacteria in solution are capable of degrading 2,4-D in solution, and that sorbed bacteria are not capable of degrading either sorbed or solution 2,4-D. Model 2 stated that only bacteria in the solution phase degrade 2,4-D in solution and that only sorbed bacteria degrade sorbed 2,4-D. Model 3 proposed that sorbed 2,4-D is completely protected from degradation and that both sorbed and solution bacteria are capable of degrading 2,4-D in solution. These models were tested by a series of controlled laboratory experiments. Models 1 and 2 did not describe the data satisfactorily and were rejected. Model 3 described the experimental results quite well, indicating that sorbed 2,4-D was completely protected from biological degradation and that sorbed- and solution-phase bacteria degraded solution-phase 2,4-D with almost equal efficiencies.     

Effects of sorption on biological degradation rates of (2,4-dichlorophenoxy) acetic acid in soils

Three mathematical models were proposed to describe the effects of sorption of both bacteria and the herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) on the biological degradation rates of 2,4-D in soils. Model 1 assumed that sorbed 2,4-D is not degraded, that only bacteria in solution are capable of degrading 2,4-D in solution, and that sorbed bacteria are not capable of degrading either sorbed or solution 2,4-D. Model 2 stated that only bacteria in the solution phase degrade 2,4-D in solution and that only sorbed bacteria degrade sorbed 2,4-D. Model 3 proposed that sorbed 2,4-D is completely protected from degradation and that both sorbed and solution bacteria are capable of degrading 2,4-D in solution. These models were tested by a series of controlled laboratory experiments. Models 1 and 2 did not describe the data satisfactorily and were rejected. Model 3 described the experimental results quite well, indicating that sorbed 2,4-D was completely protected from biological degradation and that sorbed- and solution-phase bacteria degraded solution-phase 2,4-D with almost equal efficiencies.     

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20 mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12 h dark/12 h light (600 mol photons/m²s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.This revised version was published online in November 2004 with corrections to Volume 48.     

Effects of phenoxy acid herbicides and glyphosate on nitrogenase activity (acetylene reduction) in root-associated Azospirillum, Enterobacter and Klebsiella

Abstract Nitrogenase activity (C₂H₂ reduction) in root-associated Azospirillum lipoferum, Klebsiella pneumoniae, Enterobacter agglomerans and Pseudomonas sp. isolated from roots of Finnish grasses was assayed in the presence of glyphosate, the phenoxy acid herbicides 2-methyl-4-chlorophenoxy acetic acid (MCPA), 2,4-dichlorophenoxy acetic acid (2,4-D), (±)-2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) and (±)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop), and the commercial products Roundup, Nurmikko-Hedonal, Mepro, and Dipro. In the presence of the phenoxy acid herbicides the nitrogenase activity of K. pneumoniae was significantly inhibited, but that of E. agglomerans was stimulated. With the exception of Mepro and mecoprop no phenoxy acid herbicides inhibited the nitrogenase activity of A. lipoferum and none that of Pseudomonas sp. Nurmikko-Hedonal considerably stimulated the nitrogenase activity of E. agglomerans , and Pseudomanas sp. On the other hand, the nitrogenase activity of both K. pneumoniae and E. agglomerans was considerably repressed by glyphosate and Roundup, which also inhibited the growth of the bacteria. These chemicals had no effect on the growth of A. lipoferum and Pseudomonas sp., but stimulated their nitrogenase activity.     

Effect of phenoxyalkanoic acid herbicides on the fermentative production of l-lysine

Summary The effect of the herbicides MCPA, MCPB, mecoprop, dichlorprop, 2,4-D, 2,4-DB, and 2,4,5-T on l-lysine fermentation was investigated using a lysine-producing mutant of Corynebacterium glutamicum. Stimulation of l-lysine production by 6% to 36% was observed in shaken flask experiments when the test herbicides were added at a concentration of 5 · 10^-4 M to growing cultures after 24 h of cultivation. The most effective stimulators were MCPA, mecoprop and dichlorprop.Detailed studies of the effect of MCPA (5 · 10^-6 M to 5 · 10^-3 M) showed that the degree of stimulation depended on medium composition and aeration. In the synthetic medium, maximum production of 50 g · l^-1 lys · HCl occurred at 5 · 10^-4 M MCPA and an oxygen transfer rate (OTR) of 1.97 g O₂ · l^-1 · h^-1, while 61.7 g · l^-1 of lys · HCL was formed at 5 · 10^-3 M MCPA and an OTR of 3.75 g O₂ · l^-1 · h^-1. In the amino-nitrogen rich medium, maximum production of 42 g · l^-1 lys · HCl was observed at 5 · 10^-6 M MCPA and an oxygen transfer rate of 1.5 g O₂ · l^-1 · h^-1. Results from batch l-lysine fermentation in a fermenter showed similar stimulatory effects, with an optimal concentration of MCPA for l-lysine production of 5 · 10^-5 M. Without herbicide addition, the test strain produced 16.25 g · l^-1 of product and with addition of 5 · 10^-5 M MCPA, the same strain produced 52.1 g · l^-1 lys · HCl after 72 h of fermentation.Abbreviations MCPA 2-methyl-4-chlorophenoxyacetic acid - MCPB 2-methyl-4-chlorophenoxybutyric acid - mecoprop 2-methyl-4-chlorophenoxypropionic acid - dichlorprop 2,4-dichlorophenoxypropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4-DB 2,4-dichlorophenoxybutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

Alpha-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and alpha-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the alpha-ketoglutarate-dependent dioxygenases and share the specific motif HXDX(24)TX(131)HX(10)R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 microM for (R)-mecoprop, 164 microM for (R)-dichlorprop, and 3 microM for alpha-ketoglutarate for RdpA and 132 microM for (S)-mecoprop, 495 microM for (S)-dichlorprop, and 20 microM for alpha-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 microM for SdpA and >230 microM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace alpha-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAalpha-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

Influence of redox potential on the anaerobic biotransformation of nitrogen-heterocyclic compounds in anoxic freshwater sediments

The potential for degradation of four nitrogen-heterocyclic compounds was investigated in fresh-water sediment slurries maintained under denitrifying, sulfate-reducing, and methanogenic conditions. Pyridine (10 mg/l) was rapidly transformed within 4 weeks under denitrifying conditions but persisted for up to 3 months under sulfate-reducing and methanogenic conditions. No intermediate biotransformation products of pyridine metabolism were detected under denitrifying conditions. Quinoline (10 mg/l) was completely transformed without a lag phase under methanogenic and sulfate-reducing conditions after incubation for 23 and 45 days, respectively. 2-Hydroxyquinoline was produced concomitantly with quinoline transformation under methanogenic and sulfate-reducing conditions. Under denitrifying conditions, less than 23% of the initial concentration of quinoline was transformed after anaerobic incubation for 83 days. Indole, however, was completely removed from sediment slurries under denitrifying, sulfate-reducing, and methanogenic conditions after anaerobic incubation for 18, 27, and 17 days, respectively. Only low amounts of oxindole (2–4 mg/l) accumulated during indole metabolism under methanogenic and denitrifying conditions, but under sulfate-reducing conditions, oxindole accumulation was stoichiometric with indole transformation. No evidence for biotransformation of carbazole was noted for all anaerobic conditions tested.     

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2, 4–dichlorophenoxyacetate

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.     

Effect of several herbicides on bacterial populations and activity and the persistence of these herbicides in soil

Summary Samples of a sandy loam soil were supplied with normal, 10-fold and 100-fold rates of ioxynil, dalapon, mecoprop, dichlorprop, MCPA + dichlorprop, picloram, and amitrole-T and incubated at 29°C at 65 per cent of the waterholding capacity. Treated soil samples were compared with untreated samples. Samples supplied with (NH₄)₂SO₄ and herbicides were used to investigate the effect of the herbicides on the rate of nitrification and the production of nitrite. In several cases higher numbers of bacteria were found for a longer or shorter period in soil treated with herbicides. There was some evidence that certain groups of bacteria had adapted to ioxynil in a soil sample supplied with the 100-fold rate of this chemical. After 2 or 4 weeks lower numbers ofAzotobacter chroococcum were found at the normal rate of ioxynil, dalapon, mecoprop, and dichlorprop. At the 100-fold rate of application the numbers of Azotobacter were unfavourably affected by all herbicides. The production of mineral nitrogen was hardly affected by the normal and 10-fold rates of application. In the first week the rate of nitrification was slightly depressed in soil samples treated with the normal rates of dichlorprop and amitrole-T and with the 10-fold rates of dalapon, mecoprop, and MCPA + dichlorprop. Strong inhibition of the nitrification for at least 7 weeks was shown by the 10-fold rate of amitrole-T. At the 100-fold rate all herbicides, with the exception of picloram, depressed the rate of nitrification for a longer or shorter period. During the second week a very small increase of nitrite was found in the samples treated with the normal and 10-fold rates of dalapon, mecoprop, dichlorprop, and amitrole-T. A small increase of nitrite was noted for 26 weeks in samples treated with the 100-fold rates of amitrole-T. A highly significant depression of CO₂ evolution was found in the first week in samples treated with the normal rates of ioxynil, dalapon, mecroprop, dichlorprop, and amitrole-T, also in samples treated with the 10-fold rates of dalapon, mecoprop, dichlorprop, MCPA + dichlorprop, picloram, and amitrole-T. A highly significant depression of CO₂ production was found after 8 weeks in all the samples treated with herbicides at the 100-fold rate with the exception of the sample treated with picloram. The decomposition of the herbicides was studied in soil samples treated with the 100-fold rates of herbicides. Only traces of dalapon and mecoprop were found after 9 months, but 7.2% ioxynil, 29.8% dichlorprop, 39% (MCPA + dichlorprop), 52.1% picloram and 52.2% amitrole-T were still present in active form.     

Natural Biological Attenuation of Phenoxy Herbicides in Groundwater: Dow AgroSciences Paritutu Site, New Zealand

Groundwater beneath a manufacturing site previously used for herbicide production has been shown to contain low levels of chlorinated phenols and phenoxy herbicides. The importance of biological processes in the natural attenuation of the groundwater contaminants was examined as part of an ongoing investigation. Analysis of the groundwater chemistry indicated that the aquifer is essentially aerobic in the area of interest. Laboratory microcosm experiments demonstrated that the naturally occurring microorganisms rapidly degraded a mixture of the predominant organic contaminants under conditions that simulate those in the aquifer. The time required for 50% degradation ranged from 7 to 27 days for 2,4-dichlorophenoxyacetic acid (2,4-D) and 9 to 49 days for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The rapid biodegradation rates were consistent with the results of microbiological analyses, which demonstrated that a substantial proportion of the culturable bacteria were capable of growth on 2,4-D as a sole carbon source. Results of gene probe assays suggested the numbers of bacteria with the potential to degrade 2,4-D were one to two orders of magnitude higher than were detected using plate counts. Computer model simulations illustrated that biodegradation would be expected to significantly contribute to the attenuation of 2,4-D and 2,4,5-T in the aquifer. On the basis of the various lines of evidence and the distances the groundwater must travel, the groundwater contaminants would be expected to naturally biodegrade to below levels of concern before the plume reaches potential environmental receptors.