Reaction of soil microflora to propanide treatment]

Propanide, a herbicide, is hydrolyzed in the soil into 3,4-dichloroaniline and propionic acid. The amount of microorganisms resistant to propanide and 3,4-dichloroaniline increases when the herbicide is added to the soil, and then decreases when these compounds disappear from the soil.     

Degradation of chloroanilines in soil slurry by specialized organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichloroaniline was examined as single compounds as well as a mixture in soil slurries. At 30°C the degradation of chloroanilines by indigenous soil populations in soil slurries was observed when soil slurry was freshly contaminated or precontaminated to allow binding of chloroanilines to the soil matrix. Within 6 weeks, 3-chloro- and 3,4-dichloroaniline (each 2 mm) were degraded more rapidly (about 50% chloride elimination) than 4-chloro- and 2-chloroaniline, due to stronger adsorption of 4-chloroaniline and greater resistance of 2-chloroaniline. The addition of various supplements such as buffer, mineral salts and acetate only slightly influenced the degradation of chloroanilines by the indigenous soil populations. The mineralization was drastically enhanced when laboratory-selected chloroaniline-degraders (8·10⁶ cells/g) such as Pseudomonas acidovorans strain BN3.1 were supplemented to the soil slurries so that complete elimination of chloride from the chloroanilines occurred within 10 days. Correspondence to: F. R. Brunsbach     

Degradation of mixtures of chloroaromatic compounds in soil slurry by mixed cultures of specialized organisms

The degradation of a mixture of 13 chloroaromatics, 2-chloro-, 3-chloro-, 4-chloro- and 3,4-dichloroaniline,2-chloro-, 3-chloro-,4-chloro-, 3,4-dichloro-and 3,5-dichlorobenzoate, and chloro-,1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene in soil slurries by a mixed culture of Pseudomonas acidovorans strain BN 3.1, Pseudomonas ruhlandii strain FRB2, Pseudomonas cepacia strain JH230 and Pseudomonas aeruginosa strain RHO1 was studied. About 70% of the organic bound chlorine was eliminated after 25 days from soil with a carbon content of 8% (soil 1) when 2–3 × 10⁵ cells/g soil of each of the strains were added to the slurries. The effect of the clean-up was demonstrated by a biological test using cress and wheat. Both plants showed good germination and growth on both non-contaminated soils and the contaminated soil 1 after the biotreatment with the strains. No growth was observed when the plants were incubated with the contaminated soil 1 and with the contaminated and biotreated soil 2 (carbon content 2.6%). This indicates that the remaining 30% of organic chlorine in soil 1 after biotreatment does not influence the germination and growth of the two plants tested. *** DIRECT SUPPORT *** AG903062 00010     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

Human B-cell proliferation in response to recombinant interleukin 2 is not due to T-cell help

Positively selected human B-cell suspensions with no detectable T cells and containing more than 99.5% B cells both at the initiation and termination of culture were shown to proliferate in response to interleukin 2 (IL-2) in a dose-dependent fashion. The lack of influence of residual T cells on this proliferative response was demonstrated in experiments where T cells were added back in increasing numbers to B-cell suspensions. No detectable enhancing effect on B-cell proliferation was noted when 2.5% T cells were purposely added back to culture, a proportion far in excess of that which might be expected to contaminate B-cell suspensions under the present methodology. In contrast, when 10% T cells were added back to B-cell cultures, an enhanced proliferation of B cells was observed suggesting that the lack of effect of lower numbers of T cells was due to their inefficiency in helping B-cell proliferation in response to IL-2. Therefore, it is concluded that highly purified IL-2 is capable of triggering human peripheral blood B cells to proliferate and that this proliferation is not due to T-cell help.     

3,4-dichloroaniline cometabolism by representatives of the genus Pseudomonas

The effect of propanide, linuron and 3,4-dichloroaniline on soil organisms was studied. Two strains of Pseudomonas aurantiaca 1 and 7, were isolated from soil; they decomposed propanide yielding 3,4-dichloroaniline. These strains, as well as a number of collection cultures belonging to the Pseudomonas genus, could transform 3,4-dichloroaniline at a rate of 0-100 per cent during 48 hours. A certain correlation existed between this transformation ability and the level of total oxidase activity. All the strains of Pseudomonas studied in this work were characterized by a low peroxidase activity, and no strict correlation was detected between its level and the ability to transform 3,4-dichloroaniline.     

Reasons for possible failure of inoculation to enhance biodegradation

Pseudomonas strains capable of mineralizing 2,4-dichlorophenol (DCP) and p-nitrophenol (PNP) in culture media were isolated from soil. One DCP-metabolizing strain mineralized 1.0 and 10 micrograms of DCP but not 2.0 to 300 ng/ml in culture. When added to lake water containing 10 micrograms of DCP per ml, the bacterium did not mineralize the compound, and only after 6 days did it cause the degradation of 1.0 microgram of DCP per ml. The organism did not grow or metabolize DCP when inoculated into sterile lake water, but it multiplied in sterile lake water amended with glucose or with DCP and supplemental nutrients. Its population density declined and DCP was not mineralized when the pseudomonad was added to nonsterile sewage, but the bacterium grew in sterile DCP-amended sewage, although not causing appreciable mineralization of the test compound. Addition of the bacterium to nonsterile soil did not result in the mineralization of 10 micrograms of DCP per g, although mineralization was evident if the inoculum was added to sterile soil. A second DCP-utilizing pseudomonad failed to mineralize DCP when added to the surface of sterile soil, although activity was evident if the inoculum was mixed with the soil. A pseudomonad able to mineralize 5.0 micrograms of PNP per ml in culture did not mineralize the compound in sterile or nonsterile lake water. The bacterium destroyed PNP in sterile sewage and enhanced PNP mineralization in nonsterile sewage. When added to the surface of sterile soil, the bacterium mineralized little of the PNP present at 5.0 micrograms/g, but it was active if mixed well with the sterile soil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reasons for possible failure of inoculation to enhance biodegradation.

Pseudomonas strains capable of mineralizing 2,4-dichlorophenol (DCP) and p-nitrophenol (PNP) in culture media were isolated from soil. One DCP-metabolizing strain mineralized 1.0 and 10 micrograms of DCP but not 2.0 to 300 ng/ml in culture. When added to lake water containing 10 micrograms of DCP per ml, the bacterium did not mineralize the compound, and only after 6 days did it cause the degradation of 1.0 microgram of DCP per ml. The organism did not grow or metabolize DCP when inoculated into sterile lake water, but it multiplied in sterile lake water amended with glucose or with DCP and supplemental nutrients. Its population density declined and DCP was not mineralized when the pseudomonad was added to nonsterile sewage, but the bacterium grew in sterile DCP-amended sewage, although not causing appreciable mineralization of the test compound. Addition of the bacterium to nonsterile soil did not result in the mineralization of 10 micrograms of DCP per g, although mineralization was evident if the inoculum was added to sterile soil. A second DCP-utilizing pseudomonad failed to mineralize DCP when added to the surface of sterile soil, although activity was evident if the inoculum was mixed with the soil. A pseudomonad able to mineralize 5.0 micrograms of PNP per ml in culture did not mineralize the compound in sterile or nonsterile lake water. The bacterium destroyed PNP in sterile sewage and enhanced PNP mineralization in nonsterile sewage. When added to the surface of sterile soil, the bacterium mineralized little of the PNP present at 5.0 micrograms/g, but it was active if mixed well with the sterile soil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

The effects of calcium and magnesium on water and ionic permeabilities in the sea water adapted eel,Anguilla anguilla L.

Summary The permeability characteristics to water and ions, of the seawater adapted eel, have been studied in the absence and presence of external calcium and magnesium.The absence of these divalent ions from the external medium produces an increase in the unidirectional and net water fluxes as well as a decrease in temperature coefficients. Similarly, an increase in the effluxes of sodium and chloride and a large increase in the passive loss of these ions was observed when fish were transferred to deionized water. The stimulating effect of externally added (10 mM) potassium on the sodium and chloride effluxes, when fish are transferred to fresh water is abolished in the absence of calcium and magnesium (Fig. 1). These kinetic changes are accompanied by ultrastructural changes concerned principally with the chloride cells (Figs. 2 and 3).The results are discussed in terms of the possible role of calcium and magnesium in the maintenance of the hydro-mineral equilibrium of the eel.     

N-malonyltransferases from peanut

Three distinct N-malonyltransferases were purified from peanut seedlings, accepting either anthranilic acid, D-tryptophan, or 3,4-dichloroaniline, respectively, as a substrate. Partially purified malonyl-CoA:D-tryptophan malonyltransferase also catalyzed the formation of the corresponding malonic acid conjugate when 1-aminocyclopropane-1-carboxylic acid was employed as a substrate. These N-malonyltransferases were clearly distinguished from several O-malonyltransferase activities also present in the same seedlings. N-Malonic acid conjugates have been previously isolated from peanut either as a natural constituent or after feeding with xenobiotics. By analogy to the results reported with cultured parsley cells, multiple malonyltransferases in peanut may have a role in vacuolar transport. Crude extracts of young peanut seedlings were incapable of hydrolyzing the respective N-malonic acid conjugates. However, dialyzed extracts of older plants released malonic acid from malonyl-1-aminocyclopropane-1-carboxylic acid but not from malonyl-3,4-dichloroaniline, suggesting that some N-malonic acid conjugates may be metabolized in plants which are approaching senescence.     

Biodegradation of 3,4-dichloroaniline in a fluidized bed bioreactor and a steady-state biofilm Kinetic model

Mixed culture of microorganisms immobilized onto Celite diatomaceous earth particles were used to degrade 3,4-dichloroaniline (34DCA) in a three-phase draft tube fluidized bed bioreactor. Biodegradation was confirmed as the dominant removal mechanism by measurements of the concomitant chloride ion evolution. Degradation efficiencies of 95% were obtained at a reactor retention time of 1.25 h. A mathematical model was used to describe the simultaneous diffusion and reaction of 34DCA and oxygen in the biofilms on the particles in the reactor. The parameters describing freely suspended cell growth on 34DCA were obtained in batch experiments. The model was found to describe the system well for three out of four steady states and to predict qualitatively the experimentally observed transition in the biofilm kinetics from 34DCA to oxygen limitation.     

Residual effects of 3,4-dichloroaniline on offspring born to Ceriodaphnia cf. dubia exposed for multiple generations

One of the acknowledged limitations ofconventional toxicity tests is their inabilityto evaluate the impact of toxicants onsubsequent generations. Given their relativelyshort lifespan, cladocerans in the field may beexposed to toxicants for several generations.However, it is unclear what effect such anexposure regime could have on the cladoceranfitness after removal of the toxicant. Thispaper aimed to determine the offspring fitnessof juveniles produced by adult Ceriodaphnia cf. dubia exposed toresidual levels of 3,4-dichloroaniline overfour generations. Mass cultures of Ceriodaphnia cf. dubia were maintainedfor several generations in variousconcentrations of 3,4-dichoroaniline (0, 2.5,5, 10, 15 and 20 µg/L). The mass cultureswere re-established every generation using4th brood neonates <24 h old. Eachgeneration, 4th brood neonates <24 h oldwere also transferred individually intotoxicant-free water and examined until theproduction of their 3rd brood. F1offspring of mothers exposed to 15 and20 µg/L 3,4-dichloroaniline showedsignificantly (p < 0.05) reducedreproduction compared to the controls. Nosignificant (p > 0.05) changes inreproduction due to 3,4-dichloroaniline wereobserved for the F2 and F3 offspring. However,F4 offspring of mothers exposed to all3,4-dichloroaniline concentrations showedsignificantly (p < 0.05) increasedreproduction compared to the controls. Possibleexplanations for the varying influence ofmaternal 3,4-dichloroaniline exposure on thereproductive output of the offspring arediscussed.     

The influence of Bacillus subtilis RB14-C on the development of Rhizoctonia solani and indigenous microorganisms in the soil

The effect of soil inoculation with an antagonistic strain Bacillus subtilis RB14-C on the development of Rhizoctonia solani and changes occurring in soil and rhizosphere microbial communities were studied. RB14-C was added to the soil as a water suspension of the cells or as a broth culture. Application of cell suspensions to non-planted soil reduced the number of culturable bacteria. The density of R. solani and the number of filamentous fungi were not significantly affected by RB14-C. A similar effect was observed in the rhizosphere of tomato plants growns in bacterized soil. Broth cultures of RB14-C suppressed R. solani 1 d after inoculation, but after 3 d there was no difference in the pathogen density between soil amended with broth culture and control soil. In microcosm studies, cell suspensions of RB14-C also did not inhibit growth of R. solani on filters buried in soil. However, an inhibitory effect was obtained when a broth culture of the bacterium was used. The effect of RB14-C on fungal biomass was also estimated by measurement of ergosterol concentration in soil. It was found that ergosterol was mostly derived from R. solani and that there were no significant differences in its content between untreated soil and soil treated with RB14-C. The results suggest that suppression of Rhizoctonia damping-off by B. subtilis RB14-C probably is not related to the reduction of the pathogen population in the soil.     

INGESTION AND RETENTION OF CHROOMONAS SPP. (CRYPTOPHYCEAE) BY GYMNODINIUM ACIDOTUM (DINOPHYCEAE)1

Gymnodinium acidotum Nygaard, a blue-green dinoflagellate previously shown to contain cryptophycean chloroplasts and other organelles, was observed from water and soil samples and in culture. The dinoflagellate excysts from soil samples as a mononucleated colorless cell that is positively phototactic. Colorless cells in unialgal culture remain colorless and can only be maintained less than one week whereas pigmented cells cultured unialgally grow for 10 days but then decline rapidly. Colorless cells cultured with Chroomonas spp. regain chloroplasts and have been maintained in mixed cultures for nine months. Fifty-seven percent of the dinoflagellates from mixed cultures are bi-nucleated, and three cells have been observed possibly ingesting cryptophytes. We suggest that cryptophycean chloroplasts are retained and possibly utilized by G. acidotum for at least ten days and then digested.     

Degradation of 3,4-dichloroaniline in synthetic and industrially produced wastewaters by mixed cultures freely suspended and immobilized in a packed-bed reactor

Summary Degradation of 3,4-dichloroaniline (34DCA) in aqueous by undefined cultures of free and immobilized cells was examined. Batch cultures of freely suspended cells and continuous degradation in a packed-bed reactor were studied using both synthetically concocted and industrially produced waste-waters. 34DCA was found to be degraded with a concomitant evolution of chloride ions into the bulk medium. The [acked bed reactor with biomass immobilized on celite diatomaceous earth was found to be capable of degrading over 98% of the 34DCA present in a synthetically concocted inlet stream at a concentration of 250 mg l^–1. Residence times of less than 4 h were employed, giving an overall volumetric degradation rate for the packed bed of 90 mg l^–1 h^–1. The industrially produced wastewater contained, in addition to 34DCA, aniline, 4-chloroaniline, 2,3-dichloroaniline (23DCA) and 3,4-dichloronitrobenzene. The biomass enriched on the synthetic 34DCA waste-water was found to be capable of degrading these compounds in addition to 34DCA with the exception of 23DCA. 34DCA degradation efficiencies of over 95% were obtained for the industrial waste-water at reactor residence times of 4.6 h, giving volumetric degradation rates of 24 mg l^–1 h^–1. Offprint requests to: A. G. Livingston     

Electrooptical properties of soil nitrogen-fixing bacterium Azospirillum brasilense: effect of copper ions

The effects of copper ions on the uptake of some essential metals in the biomass and the electrooptical properties of cell suspensions of the nitrogen-fixing soil bacterium Azospirillum brasilense sp. 245 were studied. Copper cations were shown to be effectively taken up by the cell biomass from the culture medium. The addition of copper ions increased the rate of uptake of some other metals present in the culture medium. This was accompanied by changes in the electrooptical characteristics of cell suspension as measured within the orienting electric field frequency range of 10 to 10,000 kHz. The effects observed during short-term incubation of A. brasilense in the presence of copper cations were less significant than during long-term incubation. These results can be used for rapid screening of microbial cultures for enhanced efficiency of sorption and uptake of metals.     

Relationship between the regulation of membrane enzyme activities by gangliosides and a possible ganglioside segregation in membrane microdomains

Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.     

Counting viableAzotobacter chroococcum in vertisols

Methods for preparing soil suspensions for countingAzotobacter chroococcum in vertisols by soil dilution and plating were investigated. Mechanical methods to promote disaggregation of soil and Azotobacter microcolonies by shaking soil suspensions with glass beads (10% w/v) or coarse sand (1–2 mm fraction) increased soil dispersion and Azotobacter colony counts. Chemical methods for disaggregation were unsatis-factory. The non-ionic detergent Agral (0.004, 0.02, 0.1, 0.5 and 2.5% w/v) had no significant effect on soil dispersion and Azotobacter count. Both sodium pyrophosphate (0.03, 0.1, 0.3 and 0.9% w/v) and sodium metaphosphate as Calgon (0.022, 0.066, 0.2, 0.6 and 1.8% w/v) increased soil dispersion but were toxic to Azotobacter. Increasing time of shaking soil: distilled water suspensions increased deflocculation of the clay and Azotobacter counts to a maximum after 6–23 hours shaking. Comparable results were obtained within 30–60 minutes of shaking with coarse sand, but shaking with coarse sand beyond 2 hours reduced counts through mechanical damage to cells. Counts from suspensions in physiological saline (0.75% NaCl) and in distilled water were similar. Counts from suspensions in Jensen's mineral base shaken for <3 hrs were lower than from distilled water due to flocculation fo the soil by^Ca2+ ions, but were higher on extended shaking up to 23 hours due to better cell protection. Shaking soil suspensions in distilled water with 10% w/v coarse sand for 30 minutes is recommended when counting Azotobacter in vertisols.