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.     

Uptake Kinetics of 2,4-Dichlorophenoxyacetate by Delftia acidovorans MC1 and Derivative Strains: Complex Characteristics in Response to pH and Growth Substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20–100 μM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 μM 2,4-D at either pH, and amounted to 15–20 nmol/min*mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min*mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.     

Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium

Summary The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h^–1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methyphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10⁸) of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol. Offprint requests to: F. Streichsbier     

Uptake kinetics of 2,4-dichlorophenoxyacetate by Delftia acidovorans MC1 and derivative strains: complex characteristics in response to pH and growth substrate

The present investigation showed that active processes were involved in the uptake of 2,4-dichlorophenoxyacetate (2,4-D) by Delftia acidovorans MC1. With 2,4-D-grown cells, uptake at pH 6.8 was highly affine and showed a complex pattern-forming intermediary plateau at 20-100 microM 2,4-D. The kinetics became increasingly sigmoidal with raising of the pH to 7.5 and 8.5, and complexity disappeared. The apparent maximum was obtained at around 400 microM 2,4-D at either pH, and amounted to 15-20 nmol/min x mg protein. Higher substrate concentrations resulted in significant inhibition. With cells grown on (RS)-2-(2,4-dichlorophenoxy)propionate, 2,4-D uptake increased significantly and reached 45 nmol/min x mg, hinting at induction of a specific carrier(s). The kinetic characteristics made it apparent that several proteins contribute to 2,4-D uptake in MC1. An open reading frame was detected which has similarity to genes encoding major facilitator superfamily (MFS) transporters. Mutant strains that lacked this gene showed altered kinetics with decreased affinity to 2,4-D at pH 6.8. A mutant with complete deficiency in phenoxyalkanoate utilization showed an almost linear uptake pattern hinting at sole diffusion. Cloning of tfdK encoding a specific transporter for 2,4-D resulted in an increased uptake rate and, above all, higher affinity at slightly alkaline conditions due to hyperbolic kinetics. The presence of carbonylcyanide m-chlorophenylhydrazone led to the subsequent strong inhibition of 2,4-D uptake, suggesting proton symport as the likely active mechanism.     

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.     

Adaptation of Delftia acidovorans for degradation of 2,4-dichlorophenoxyacetate in a microfluidic porous medium

Delftia acidovorans MC1071 can productively degrade R-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP) but not 2,4-dichlorophenoxyacetate (2,4-D) herbicides. This work demonstrates adaptation of MC1071 to degrade 2,4-D in a model two-dimensional porous medium (referred to here as a micromodel). Adaptation for 2,4-D degradation in the 2 cm-long micromodel occurred within 35 days of exposure to 2,4-D, as documented by substrate removal. The amount of 2,4-D degradation in the adapted cultures in two replicate micromodels (~10 and 20 % over 142 days) was higher than a theoretical maximum (4 %) predicted using published numerical simulation methods, assuming instantaneous biodegradation and a transverse dispersion coefficient obtained for the same pore structure without biomass present. This suggests that the presence of biomass enhances substrate mixing. Additional evidence for adaptation was provided by operation without R-2,4-DP, where degradation of 2,4-D slowly decreased over 20 days, but was restored almost immediately when R-2,4-DP was again provided. Compared to suspended growth systems, the micromodel system retained the ability to degrade 2,4-D longer in the absence of R-2,4-DP, suggesting slower responses and greater resilience to fluctuations in substrates might be expected in the soil environment than in a chemostat.     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

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.     

Growth Rates of a Pseudomonad on 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

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.     

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.     

Continuous biodegradation of phenoxyalkanoate herbicides by Sphingomonas herbicidovorans MH in a PU-supplied bubble reactor

The continuous aerobic degradation of phenoxyalkanoate herbicides by Sphingomonas herbicidovorans MH was investigated in a bubble reactor filled with modified polyurethane-foam (PU 90/51) as a carrier for the adsorptive immobilization of the bacterial cells. The PU-foam was applied in the form of plates (5 × 10 × 10 mm) and the amount added was equivalent to a PU-load of 1.25% [w/v]. Strain MH is capable of detoxifying the dichloro-substituted phenoxyalkanoates 2,4-DP, 2,4-D and 2,4-DB and the methylchloro-substituted phenoxyalkanoates MCPA, MCPP and MCPB. Degradation of the respective substrate was followed by HPLC analyses and by determination of the chloride release. No intermediates of the degradation pathways or “dead end” products were detected by HPLC analyses. The PU-bubble reactor with immobilized 2,4-DP-pre-grown cells was run continuously at 30°C at the high dilution rate of D = 0.5h^?1 with 2,4-DP (0.2 g/l), and with subsequent changes to each of the other phenoxyalkanoates as a single substrate in the feed and with an intermittent return to 2,4-DP. Finally, after an intermediate substrate accumulation, 2,4-D, 2,4-DP, MCPA and MCPP could be degraded under the aforementioned conditions corresponding to a maximum degradation rate of Q_phen = 100 mg/l × h. In the case of 2,4-DB, a slightly reduced conversion rate of about 94% could be calculated. In contrast to these results, 0.2 g/l of the more recalcitrant MCPB could not be metabolized at this high dilution rate of D = 0.5 h^?1 by the biofilm of Sphingomonas herbicidovorans MH, but it was degradable at a reduced dilution rate of D = 0.25 h^?1. Complete detoxification of a stoichiometric mixture of the dichloro- and the methylchloro-substituted phenoxyalkanoates including MCPB, respectively, at a total concentration of 0.2 g/l was achieved at D = 0.25 h^?1, corresponding to a degradation rate of Q_tot = 50 mg/l × h. Finally, the efficiency of the PU-immobilized cells of Sphingomonas herbicidovorans MH in detoxifying mixtures of all six herbicides could be increased to Q_tot = 75 mg/l × h by the further addition of PU-foam particles corresponding to a final PU-load of 2.5% [w/v]. This PU-bubble reactor was successfully operated for more than 12 months to clean up synthetically concocted waste waters with fluctuations in phenoxyalkanoate concentration and composition.     

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.     

Metabolism of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Soybean Root Callus : EVIDENCE FOR THE CONVERSION OF 2,4-D AMINO ACID CONJUGATES TO FREE 2,4-D

An auxin-requiring soybean root callus metabolized [1-¹⁴C]-2,4-dichlorophenoxyacetic acid (2,4-D) to diethyl ether-soluble amino acid conjugates and water-soluble metabolites. The uptake in tissue varied with incubation time, concentration, and amount of tissue. Uptake was essentially complete (80%) after a 24-hour incubation and the percentage of free 2,4-D in the tissue fell to its lowest point at this time. At later times, the percentage of free 2,4-D increased and the percentage of amino acid conjugates decreased, whereas the percentage of water-soluble metabolites increased only slightly. Similar trends were seen if the tissue was incubated for 24 hours in radioactive 2,4-D, followed by incubation in media without 2,4-D for 24 hours. Inclusion of nonlabeled 2,4-D during the 24-hour chase period did not reduce amino acid conjugate disappearance but did reduce the percentage of free [1-¹⁴C]2,4-D. Thus, an external supply of 2,4-D does not directly prevent amino acid conjugate metabolism in this tissue. It is concluded that 2,4-D amino acid conjugates were actively metabolized by this tissue to free 2,4-D and water-soluble metabolites.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Morphogenetic effects of 2,4-dichlorophenoxyacetic acid on pinto bean (Phaseolus vulgaris L.) leaf explants in vitro

Roots, callus and/or globular structures were produced on primary leaf and distal cotyledon explants of pinto bean (Phaseolus vulgaris L. cv. UI 114) cultured on semisolid MS medium with a wide range of 2,4-D concentrations (0.01 to 80 mg/L) with either 0 or 1.0 mg/L kinetin. Explants rooted at lower 2,4-D concentrations than at those favoring globule formation on callus, although roots, callus and globules often developed from the same explant. Isolated opaque green globular structures developed when callus initiated on media with 3 or more mg/L 2,4-D was subcultured in liquid MS + 30 mg/L 2,4-D. These structures multiplied with a fresh weight doubling time of 8–9 days in MS + 30 mg/L 2,4-D. Although this multiplicative behavior and opaque color were reminiscent of embryoids reported for other species, no cotyledons or roots were seen.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - MS Murashige-Skoog medium Cooperative investigations of the Agricultural Research Service, U.S. Department of Agriculture and the Michigan Agricultural Experiment Station, East Lansing, Michigan 48824. Michigan Agricultural Experiment Station Journal article No. 11923     

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.     

Somatic embryogenesis inGnetum ula Brongn. (Gnetum edule) (Willd) Blume

Summary Somatic embryos ofGnetum ula (Gnetum edule) an endangered gymnosperm closely related to the angiosperms have been induced in vitro. Megagametophyte tissue with immature embryos was cultured on Murashige and Skoog medium. A mucilaginous, translucent embryogenic callus was obtained with 5 mg/l BA. Callus induced with 2,4-D was non-embryogenic. The embryogenic callus in liquid half strength Murashige and Skoog medium without inorganic nitrates supplemented with 2.5 g/l casein hydrolysate and 0.5 g/l L-glutamine gave rise to immature embryos. The embryos matured when treated with 60 g/l sucrose and 10 mg/l abscisic acid.Abbreviations MS Murashige and Skoog - BA 6-benzylaminopurine - 2,4-D 2,4 - dichlorophenoxyacetic acid - ABA abscisic acid     

Kinetic analysis of simultaneous 2,4-dinitrotoluene (DNT) and 2, 6-DNT biodegradation in an aerobic fluidized-bed biofilm reactor.

We previously reported on the mineralization of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in an aerobic fluidized-bed bioreactor (FBBR) (Lendenmann et al. 1998 Environ Sci Technol 32:82-87). The current study examines the kinetics of 2, 4-DNT and 2,6-DNT mineralization at increasing loading rates in the FBBR with the goal of obtaining system-independent kinetic parameters. At each steady state, the FBBR was subjected to a set of transient load experiments in which substrate flux in the biofilm and bulk substrate concentrations were measured. The pseudo-steady-state data were used to estimate the biokinetic parameters for 2,4-DNT and 2,6-DNT removal using a mechanistic mathematical biofilm model and a routine that minimized the sum of the squared residuals (RSS). Estimated kinetic parameters varied slightly for each steady-state; retrieved parameters for qm were 0. 83 to 0.98 g DNT/g XCOD d for 2,4-DNT removal and 0.14 to 0.33 g DNT/g XCOD d for 2,6-DNT removal. Ks values for 2,4-DNT removal (0. 029 to 0.36 g DNT/m3) were consistently lower than Ks values for 2, 6-DNT removal (0.21 to 0.84 g DNT/m3). A new approach was introduced to estimate the fundamental biofilm kinetic parameter S*b,min from steady-state performance information. Values of S*b,min indicated that the FBBR performance was limited by growth potential. Adequate performance of the examined FBBR technology at higher loading rates will depend on an improvement in the growth potential. The obtained kinetic parameters, qm, Ks, and S*b,min, can be used to aid in the design of aerobic FBBRs treating waters containing DNT mixtures.