Biodegradation of atrazine in surface soils and subsurface sediments collected from an agricultural research farm

The purpose of the present study was to assess atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) mineralization by indigenous microbial communities and to investigate constraints associated with atrazine biodegradation in environmental samples collected from surface soil and subsurface zones at an agricultural site in Ohio. Atrazine mineralization in soil and sediment samples was monitored as ¹⁴CO₂ evolution in biometers which were amended with ¹⁴C-labeled atrazine. Variables of interest were the position of the label ([U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine), incubation temperature (25°C and 10°C), inoculation with a previously characterized atrazine-mineralizing bacterial isolate (M91-3), and the effect of sterilization prior to inoculation. In uninoculated biometers, mineralization rate constants declined with increasing sample depth. First-order mineralization rate constants were somewhat lower for [2-¹⁴C-ethyl]-atrazine when compared to those of [U-¹⁴C-ring]-atrazine. Moreover, the total amount of ¹⁴CO₂ released was less with [2-¹⁴C-ethyl]-atrazine. Mineralization at 10°C was slow and linear. In inoculated biometers, less ¹⁴CO₂ was released in [2-¹⁴C-ethyl]-atrazine experiments as compared with [U-¹⁴C-ring]-atrazine probably as a result of assimilatory incorporation of ¹⁴C into biomass. The mineralization rate constants (k) and overall extents of mineralization (P _max ) were higher in biometers that were not sterilized prior to inoculation, suggesting that the native microbial populations in the sediments were contributing to the overall release of ¹⁴CO₂ from [U-¹⁴C-ring]-atrazine and [2-¹⁴C-ethyl]-atrazine. A positive correlation between k and aqueous phase atrazine concentrations (C _eq ) in the biometers was observed at 25°C, suggesting that sorption of atrazine influenced mineralization rates. The sorption effect on atrazine mineralization was greatly diminished at 10°C. It was concluded that sorption can limit biodegradation rates of weakly-sorbing solutes at high solid-to-solution ratios and at ambient surface temperatures if an active degrading population is present. Under vadose zone and subsurface aquifer conditions, however, low temperatures and the lack of degrading organisms are likely to be primary factors limiting the biodegradation of atrazine.     

Biodegradation of alkylpyridines by bacteria isolated from a polluted subsurface

Ten bacterial strains were isolated fromalkylpyridine polluted sediments 7.6 m below thesurface. These strains were able to degrade 11different alkylpyridine isomers. Degradation ratesdepended on number and position of the alkyl group. Isomers with an alkyl group at position 3 were moreresistant to microbial attack. Of the 10 strains, 6isolates were selected for detailed study. Theseisolates mineralized the isomers to CO₂,NH₄ ⁺, and biomass. All strains weregram-negative rods with a strict aerobic metabolism. Characterization of physiological and biochemicalproperties revealed similarity between strains. Eeachstrain however, had a limited substrate range whichenabled it to degrade no more than 2 to 3 compounds ofthe 14 alkylpyridine isomers tested. Examination ofthe genetic variability among cultures with therandomly amplified polymorphic DNA technique revealedhigh levels of genomic DNA polymorphism. The highestsimilarity between 2 strains (0.653) was observedbetween 2-picoline and 3-picoline degrading cultures. The molecular basis of the differences in substratespecificity is under investigation.     

Contribution of ethylamine degrading bacteria to atrazine degradation in soils

Bacterial communities that cooperatively degrade atrazine commonly consist of diverse species in which the genes for atrazine dechlorination and dealkylation are variously distributed among different species. Normally, the first step in degradation of atrazine involves dechlorination mediated by atzA, followed by stepwise dealkylation to yield either N-ethylammelide or N-isopropylammelide. As the liberated alkylamine moieties are constituents of many organic molecules other than atrazine, it is possible that a large number of alkylamine-degrading bacteria other than those previously described might contribute to this key step in atrazine degradation. To examine this hypothesis, we isolated 82 bacterial strains from soil by plating soil water extracts on agar media with ethylamine as a sole carbon source. Among the relatively large number of isolates, only 3 were able to degrade N-ethylammelide, and in each case were shown to carry the atzB gene and atzC genes. The isolates, identified as Rhizobium leguminosarum, Flavobacterium sp., and Arthrobacter sp., were all readily substituted into an atrazine-degrading consortium to carry out N-ethylammelide degradation. The distribution of these genes among many different species in the soil microbial population suggests that these genes are highly mobile and over time may lead to generation of various atrazine-degrading consortia.     

Effects of biochar amendment on the sorption and degradation of atrazine in different soils

ABSTRACT

Sugarcane top-derived biochar was added to an alluvial soil, a moist soil and a paddy soil at the rate of 0.2% and 0.5% (w/w). After the addition of 0.2% and 0.5% biochar, the sorption coefficients (K_d) of atrazine (Ce = 10 mg L^?1) were increased by 26.97% and 79.58%, respectively, in the moist soil with a low level of total organic carbon (TOC), while it increased by 31.43% and 60.06%, respectively, in the paddy soil with a high TOC content. The half-time persistence values of atrazine in the alluvial soil, moist soil and paddy soil were 28.18, 23.74 and 39.84 d, respectively. In the 0.2% biochar amended soils, the corresponding half-times of atrazine for the alluvial soil, moist soil and paddy soil were extended by 10.33, 11.81 and 1.42 d, and they were prolonged by 16.83, 17.52 and 14.74 d, respectively, in the 0.5% biochar amended soils. Atrazine degradation products (deisopropylatrazine and desethylatrazine) decreased after they accumulated to 3.2 and 1 mg kg^?1, respectively. Generally, increasing sorption was accompanied by decreasing degradation of atrazine which is found in biochar-amended soils.     

Effects of dissolved organic matter from sewage sludge on the atrazine sorption by soils

The effects of dissolved organic matter (DOM), water soluble organic matter derived from sewage sludge, on the sorption of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-trazine) by soils were studied using a batch equilibrium technique. Six paddy soils, chosen so as to have different organic carbon contents, were experimented in this investigation. Atrazine sorption isotherms on soils were described by the linear equation, and the distribution coefficients without DOM (Kd) or with DOM (Kd*) were obtained. Generally, the values of Kd*/Kd initially increased and decreased thereafter with increasing DOM concentrations of 0-60 mg DOC ·L-1 in soil-solution system form. Critical concentrations of DOM (DOMnp) were obtained where the value of Kd* was equal to Kd. The presence of DOM with concentrations lower than DOMnp promoted atrazine sorption on soils (Kd* > Kd), whereas the presence of DOM with concentrations higher than DOMnp tended to inhibit atrazine sorption (Kd* < Kd). Interestingly, DOMnp for     

Effects of dissolved organic matter from sewage sludge on the atrazine sorption by soils

The effects of dissolved organic matter (DOM), water soluble organic matter derived from sewage sludge, on the sorption of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-trazine) by soils were studied using a batch equilibrium technique. Six paddy soils, chosen so as to have different organic carbon contents, were experimented in this investigation. Atrazine sorption isotherms on soils were described by the linear equation, and the distribution coefficients without DOM (Kd) or with DOM (Kd*) were obtained. Generally, the values of Kd*/Kd initially insoil-solution system form. Critical concentrations of DOM (DOMnp) were obtained where the value of Kd* was equal to Kd. The presence of DOM with concentrations lower than DOMnp promoted atrazine sorption on soils (Kd* ＞ Kd), whereas the presence of DOM with concentrations higher than DOMnp tended to inhibit atrazine sorption (Kd* ＜ Kd). Interestingly, DOMnp for tested soils was negatively correlated to the soil organic carbon content, and the maximum of Kd*/Kd (i.e.Kmax) correlated positively with the maximum of DOM sorption on soil (Xmax). Further investigations showed that the presence of hydrophobic fraction of DOM evidently promoted the atrazine sorption on soils, whereas the presence of hydrophilic DOM fraction obviously tended to inhibit the atrazine sorption. Interactions of soil surfaces with DOM and its fractions were suggested to be the major processes determining atrazine sorption on soils. The results of this work provide a reference to the agricultural use of organic amendment such as sewage sludge for improving the availability of atrazine in soils.     

Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene

Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p-atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p-atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p-atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium-mediated transformation. Our work suggests that the in planta expression of p-atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome.     

Biodegradation of 4-nitrophenol by indigenous microbial populations in Everglades soils

The Everglades in South Florida are a unique ecologicalsystem. As a result of the widespread use of pesticides andherbicides in agricultural areas upstream from these wetlands,there is a serious potential for pollution problems in theEverglades. The purpose of this study was to evaluate theability of indigenous microbial populations to degradexenobiotic organic compounds introduced by agricultural andother activities. Such biodegradation may facilitate theremediation of contaminated soils and water in the Everglades.The model compound selected in this study is 4-nitrophenol, achemical commonly used in the manufacture of pesticides. Themineralization of 4-nitrophenol at various concentrations wasstudied in soils collected from the Everglades. Atconcentrations of 10 and 100 µg/g soil, considerablemineralization occurred within a week. At a higherconcentration, i.e., 10 mg/g soil, however, no mineralizationof 4-nitrophenol occurred over a 4-month period; such a highconcentration apparently produced an inhibitory effect. Therate and extent of 4-nitrophenol mineralization was enhancedon inoculation with previously isolated nitrophenol-degradingmicroorganisms. The maximum mineralization extent measured,however, was less than 30% suggesting conversion to biomassand/or unidentified intermediate products. These resultsindicate the potential for natural mechanisms to mitigate theadverse effects of xenobiotic pollutants in a complex systemsuch as the Everglades.     

Biodegradation of atrazine in sand sediments and in a sand-filter

The potential of a microbial consortium for treating waters contaminated with atrazine was considered. In conventional liquid culture, atrazine and its two dealkylated by-products were equally metabolised by the microbial consortium. Transient production of hydroxyatrazine was observed during atrazine catabolism, indicating that the catabolic pathway was similar to the one reported for isolates capable of atrazine mineralisation. This consortium was then inoculated to sediments sampled from an artificial recharge site. These sediments were contaminated by atrazine and diuron and exhibited only a slow endogenous herbicide dissipation. Inoculated microorganisms led to extensive atrazine degradation and survived for more than 10 weeks in the sediments. A rudimentary bioreactor was then setup using a soil core originating from the same recharge site. Degrading microorganisms rapidly colonised the core and expressed their degrading activity. The efficiency of the bioreactor was improved in the presence of spiked environmental surface waters. Atrazine degraders thus possibly benefited from the other organic sources in developing and expressing their activity. The microbial consortium did not initially exhibit the capacity to degrade diuron, which was used as reference compound. No change in this characteristic was detected throughout the study. Received: 13 December 1999 / Received revision: 26 April 2000 / Accepted: 5 May 2000     

Atrazine degradation by stable mixed cultures enriched from agricultural soil and their characterization

Aims:  The aim of this work was to enrich stable mixed cultures from atrazine-contaminated soil. The cultures were examined for their atrazine biodegradation efficiencies in comparison with J14a, a known atrazine-degrading strain of Agrobacterium radiobacter . The cultures were also characterized to identify community structure and bacterial species present.
Methods and Results:  The cultures were enriched and then stabilized in bacterial media. The stable mixed cultures and J14a were tested in a medium containing 100 μg l⁻¹ of atrazine. For all cultures, atrazine was removed 33–51% within 7 days and the cell optical density increased from 0·05 to between 0·50 and 0·70. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence analysis of the 16 S rRNA gene as Alcaligenes faecalis , Klebsiella ornithinolytica , Bacillus megaterium and Agrobacterium tumefaciens , respectively. An atrazine-degrading gene, atzA , was present in ND2 and ND4.
Conclusions:  The stable mixed cultures obtained could degrade atrazine. Klebsiella ornithinolytica ND2 and Ag. tumefaciens ND4 are atrazine degraders.
Significance and Impact of the Study:  The novel stable mixed cultures could be used for bioremediating crop fields contaminated with atrazine. This is the first report of the atzA gene in Kl. ornithinolytica .     

Biodegradation of benzene, toluene and naphthalene in soil-water slurry microcosms

Aerobic biodegradation of benzene, toluene andnaphthalene was studied in pre-equilibrated soil-waterslurry microcosms. The experiments were designed tosimulate biodegradation at waste sites where sorptionreaches equilibrium before biodegradation becomesimportant. Rates of biodegradation were reduced by thepresence of soil. For example, nearly completenaphthalene biodegradation (1.28 mg/L) by indigenoussoil bacteria occurred within 60 hours in aqueoussolution (soil-free) while it took two weeks todegrade the same amount in the presence of 0.47 kgsoil/L of water. The rate of biodegradation wasobserved to decrease with increasing organic compoundhydrophobicity, soil/water ratio, soil particle size,and soil organic carbon content. These resultsclearly indicate that the rate of biodegradation isaffected by both the extent and rate of sorption. Further analysis suggests that mass transfer couldcontrol the performance of in situ bioremediation forhighly hydrophobic organic contaminants which exhibita large extent of sorption and slow rate ofdesorption.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Plasmid incidence in bacteria from agricultural and industrial soils

Heavy metal contents of agricultural and industrial soils were determined by atomic absorption spectrophotometry. The analysis of the samples collected from two different locations revealed significantly high levels of Fe, Zn, Cu, Cr and Ni. Certain microbiological parameters (total aerobic heterotrophs, asymbiotic N₂-fixers, total Actinomycetes and fungi) were also monitored from these soils. A total of 70 bacterial isolates from agricultural and industrial soils were examined for plasmid DNA content and resistance to the antibiotics amoxycillin, cloxacillin, chloramphenicol, doxycycline methicillin, nalidixic acid, and tetracycline. Minimum inhibitory concentrations (MICs) of Cu, Cr, Pb, Cd, Hg, Zn, and Ni for each isolate were also determined. Resistance was most frequent to methicillin (48.5%), cloxacillin (45.7%), and nalidixic acid (40%) for all isolates of bacteria. The highest MICs observed were 100 g/ml for mercury, 800 g/ml for Ni and 1600 g/ml for other metals. The incidences of metal resistance and MICs of metals for bacteria from industrial soil were significantly different to those of agricultural soil. On a percentage basis, 91.4% of the total bacterial isolates from industrial soil were found to harbour plasmids whereas 40% of the isolates from agricultural soil contained plasmids.     

Predominance of Char Sorption over Substrate Concentration and Soil pH in Influencing Biodegradation of Benzonitrile

Incomplete combustion of field crop residues results in the production of char, a material rich in charcoal-type substances. Consequently, char is an effective adsorbent of organic compounds and when incorporated into soil may adsorb soil-applied pesticides, thereby altering their susceptibility to biodegradation. We investigated the relative importance of char, soil pH and initial substrate concentration in biodegradation of pesticides in soils by measuring the biodegradation of benzonitrile in soil as a function of soil char content (0% and 1% by weight), initial benzonitrile concentration (0.1, 1.06, and 10.2 mg l⁻¹) and soil pH (5.2, 6.9 and 8.5). Preliminary experiments revealed that wheat straw char had a much greater benzonitrile sorption capacity than did soil to which the char was added. The extent of benzonitrile degradation decreased as initial benzonitrile concentration increased in both buffer solution and soil slurry. In contrast, the degradation increased as initial benzonitrile concentration increased in char-amended slurry. In un-amended soil slurry, the benzonitrile degradation was lower at pH 5.2 than at pH 6.9 or 8.5, but in char-amended soil slurry the degradation was not affected by pH, again presumably due to adsorption of benzonitrile by the char. Adsorption by soil char appears to be more important than either initial substrate concentration or soil pH in controlling benzonitrile degradation in char-amended soil slurry. The presence of crop residue-derived chars may alter pesticide degradation patterns normally observed in soils and thus significantly affect their environmental fate.     

Modeling of DNAPL-Dissolution, Rate-Limited Sorption and Biodegradation Reactions in Groundwater Systems

This article presents an approach for modeling the dissolution process of single component dense non-aqueous phase liquids (DNAPL), such as tetrachloroethene and trichloroethene, in a biologically reactive porous medium. In the proposed approach, the overall transport processes are conceptualized as three distinct reactions. Firstly, the dissolution (or dissolving) process of a residual DNAPL source zone is conceptualized as a mass-transfer limited reaction. Secondly, the contaminants dissolved from the DNAPL source are allowed to partition between sediment and water phases through a rate-limited sorption reaction. Finally, the contaminants in the solid and liquid phases are allowed to degrade by a set of kinetic-limited biological reactions. Although all of these three reaction processes have been researched in the past, little progress has been made towards understanding the combined effects of these processes. This work provides a rigorous mathematical model for describing the coupled effects of these three fundamental reactive transport mechanisms. The model equations are then solved using the general-purpose reactive transport code RT3D (Clement, 1997).     

Biodegradation of polycyclic aromatic hydrocarbons by Sphingomonas strains isolated from the terrestrial subsurface

Several strains of Sphingomonas isolated from deep Atlantic coastal plain aquifers at the US Department of Energy Savannah River Site (SRS) near Aiken, SC were shown to degrade a variety of aromatic hydrocarbons in a liquid culture medium. Sphingomonas aromaticivorans strain B0695 was the most versatile of the five strains examined. This strain was able to degrade acenaphthene, anthracene, phenanthrene, 2,3-benzofluorene, 2-methylnaphthalene, 2,3-dimethylnaphthalene, and fluoranthene in the presence of 400 mg l⁻¹ Tween 80. Studies involving microcosms composed of aquifer sediments showed that S. aromaticivorans B0695 could degrade phenanthrene effectively in sterile sediment and could enhance the rate at which this compound was degraded in nonsterile sediment. These findings indicate that it may be feasible to carry out (or, at least, to enhance) in situ bioremediation of phenanthrene-contaminated soils and subsurface environments with S. aromaticivorans B0695. In contrast, strain B0695 was unable to degrade fluoranthene in microcosms containing aquifer sediments, even though it readily degraded this polynuclear aromatic hydrocarbon (PAH) in a defined liquid growth medium. Journal of Industrial Microbiology & Biotechnology (2001) 26, 283–289. Received 25 September 2000/ Accepted in revised form 08 February 2001     

Biodegradation of Dichloromethane in an Estuarine Environment

Dichloromethane (DCM) is a toxic pollutant showing prolonged persistence in water. So far, biodegradation of DCM has only been reported in soils and freshwater systems. Herein, we studied whether or not biodegradation of DCM could occur in estuarine waters. Results showed over 90% mineralization of DCM in natural estuarine waters supplemented with DCM. Biodegradation of DCM in estuarine waters occurred by association of different bacterial species. Generally, two bacterial species participated in DCM degradation. Two bacterial consortia were obtained. Consortia were able to degrade around 80% of DCM in about 6 days. The species involved in the process were identified by 16S rRNA gene sequencing; a consortium was constituted by Pseudomonas sp. and Brevundimonas sp. and a second consortium was formed by Pseudomonas sp. and an Acinetobacter sp. Our results showed that DCM can be readily biodegraded in estuarine waters.     

Stress induced phosphate solubilization in bacteria isolated from alkaline soils

Phosphate solubilizing bacteria NBRI0603, NBRI2601, NBRI3246 and NBRI4003 were isolated from the rhizosphere of chickpea and alkaline soils. All four strains demonstrated diverse levels of phosphate solubilization activity under in vitro conditions in the presence of various carbon and nitrogen sources. Acid production may have contributed to phosphate solubilization, but was not the only reason for phosphate release into the medium. Among the four strains, NBRI2601 was the most efficient strain in terms of its capability to solubilize phosphorus in the presence of 10% salt, pH 12, or 45 degrees C. The strains showed varied levels of phosphate solubilization when the effects of different sources of nitrogen were examined during growth. The presence of low levels of Ca(2+) and EDTA in the medium enhanced phosphate solubilization.