Amendment of organic wastes and composts to soil is a common agricultural practice. The soil microbiota is responsible for the recycling and mineralization of these organic amendments and their activity affects soil structure and agricultural viability. In this study we assessed the short-term effects of compost amendment on bacterial community composition and activity in agricultural soil. Changes in biomass, respiration and enzymatic activity were correlated with bacterial community profiles, as determined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S ribosomal RNA (rRNA) gene fragments and sequencing of relevant bands. Prior to wetting, the community profile of the compost amended soils was almost identical to that of the original compost, implying that the compost bacterial content was much higher than that of the soil. Nonetheless, wetting of compost-amended soils caused an immediate shift in the bacterial community profile resulting in a pattern similar to that of the original soil profile. Initially, in the first three days following wetting, the bacterial community profiles of the control and compost-amended soils were highly similar to each other. However, from day five to day 13, significant differences in the bacterial profiles developed between the control and compost-amended soils, indicating that the compost-related effect on the bacterial community is somewhat delayed. Compost-amended soils showed enhanced rates of nitrification, respiration, hydrolytic and metabolic activities relative to the control soils without compost. They were also characterized by proliferation of communities associated with the Bacteroidetes phylum, which may be explained by the involvement of this clade in the degradation of compost-derived complex organic substrates in the soil microcosms.

The ways in which microorganisms affect the fate of s-triazine herbicides and, in a larger sense, all organic pesticides in the environment, are the subject of intense investigation. Among the pesticides, atrazine represents one of the most extensively investigated compounds.Recently there have been several advances in our understanding of the cellular and molecular mechanism(s) by which microorganisms transform s-triazines. Enzymes and genes for the dealkylation, dechlorination, and subsequent mineralization of s-triazine herbicides to harmless compounds - such as carbon dioxide (CO2) and ammonia (NH3) - have been reported in great detail. Moreover, sequencing of these genes has led to the finding of homologous genes on transferable elements (plasmids) among various bacterial species. Some microorganisms have the ability to remove s-triazines completely from the environment. Current research is focused on the application of these fundamental results in order to develop better tools to understand microbial degradation and aid in environmental remediation.

The simultaneous removal of atrazine and nitrate in continuous culture under denitrifying conditions using Pseudomonas sp. strain ADP was investigated. Under all operational conditions the nitrate removal efficiency was always higher than 90%, while atrazine degradation deteriorated with time due to contamination by foreign denitrifying bacteria, lacking the ability to degrade atrazine. Recovery of atrazine degradation ability was achieved by applying aerobic conditions with atrazine as the sole nitrogen source.

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the alpha-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.

The potential of soil microorganisms to mineralize atrazine was studied in soil samples collected from fields with various histories of atrazine application. In contrast to many previous studies, which showed no atrazine mineralization activity, all the tested soils mineralized atrazine regardless of their atrazine application history. However, the delay before mineralization and the variation in the subsequent mineralization rate were in agreement with the initial copy number of the atrazine dechlorinaze gene, and the proliferation rate of the degraders. Soils from corn fields, which had up to 100 copies of the atzA gene per gram of soil, had a lag period of 4-5 days before atrazine mineralization started, and final mineralization percentages ranged from 40% to 54%. However, soils from fields that were never amended with atrazine had much longer lag periods (more than 17 days), which decreased after enrichment of the degrader population with high concentrations of atrazine for 15 days. Generally the mineralization rate and the atzA gene copy number increased after the enrichment period. The atrazine mineralization potential was measured by PCR of genes from the atrazine mineralization pathway. Magnetic capture hybridization was the most efficient of the two tested methods for purifying target DNA of PCR inhibitors, without reducing the copy number of the required fragment. Nested PCR proved to be the most effective method for predicting the exact potential of the soil to mineralize the pollutant even without enrichment of a small population with the target genes. This method can complement microcosm studies and eliminate futile efforts when the potential to mineralize the pollutant does not exist in the soil.

After the failure of a three-month pump-and-treat exercise to clean up an aquifer contaminated with the pesticides atrazine and fenamiphos, microcosm experiments using 14C-labeled compounds were undertaken to determine under what conditions bioremediation would be most effective, and to investigate the prospects for the use of bioaugmentation. The calculated half-lives for atrazine and fenamiphos mineralization to carbon dioxide in unamended, anaerobic aquifer material were 730 and 1,000 years, respectively. Oxygenation, coupled with bioaugmentation with enrichments of atrazine-mineralizing bacteria obtained from the contaminated site or an imported, atrazine-mineralizing pure strain, Pseudomonas sp. strain ADP, decreased the half-life of atrazine mineralization, to >20 days. Although strain ADP does not use atrazine as a source of carbon and energy, amendment of the aquifer material with citrate, which strain ADP uses as a source of carbon and energy, did not appreciably stimulate the mineralization rate of atrazine in the microcosms, suggesting that the aquifer contains enough natural organic carbon for atrazine mineralization. Aerobic enrichments of fenamiphos-degrading bacteria were prepared; however, oxygenation and bioaugmentation of aquifer material with these strains did not enhance mineralization of fenamiphos within the time constraints of the experiments. The shortest calculated half-life of fenamiphos mineralization in the microcosms was 6.8 years, which is exceedingly long compared with the half-life of fenamiphos in most surface soils.

s-Triazines including atrazine are heavily used agricultural herbicides, and their extensive removal from industrial wastewater is required before these effluents can be disposed. The use of enzymes for this purpose is an important potential alternative to conventional methods of detoxification. The purpose of the present study was to develop an enzyme-based technology for the treatment of atrazine contaminated water. In this paper we describe the construction and expression of two fusion proteins which dechlorinate atrazine while being firmly bound to an insoluble cellulose matrix. Dechlorination of atrazine produces in one enzymatic step the nonregulated compound hydroxyatrazine from the regulated mother compound, atrazine.

Indigenous soil bacteria significantly mineralized atrazine irrespective of sand depth or treatment type. After 32 d, the mineralization ranged from 0.3 to 75%, with a variable lag period before the initiation of mineralization, indicating the presence of genes for atrazine mineralization. Soil DNA extraction followed by magnetic capture hybridization-PCR revealed the presence of the genes atzA, atzB and atzC, indicating potential mineralization via the same pathway as in Pseudomonas sp. strain ADP (P.ADP). When P.ADP was inoculated into the sands, its atzA copy number declined after 1 d from the initial inoculation size (7.5×106 copies g−1 sand) by at least two orders of magnitude (<3.9×104 copies g−1 sand) with no significant recovery after 18 d. In spite of atzA low copy number in the sand, 40 and 75% atrazine mineralization occurred after 1 week when the sand was irrigated with tap water or wastewater, respectively. Amendment with composted sludge, resulted in a similar mineralization rate to that in the sands irrigated with wastewater alone, when the Kd value for atrazine was less than 1.17 l kg−1, regardless of the irrigation water quality. In two replicates of the 10–20-cm layer, with Kd values of 1.57 and 2.79 l kg−1, only 23 and 5%, respectively, of the applied atrazine was mineralized. These observations suggest that, even though sludge amendment or wastewater irrigation increased the competition between indigenous populations and introduced bacteria, P.ADP was able to continue mineralizing atrazine. The atzA copy numbers remain in the treated sand in low but stable (and active) concentrations. The high organic matter content of the sludge was the main factor affecting atrazine mineralization, because of its atrazine sorption ability.

Atrazine mineralization was studied in sediments takenfrom a shallow aquifer underlying a cornfield continuouslyreceiving atrazine and terbuthylazine. In contrast toprevious publications indicating slow or nonexistingmineralizationratesunderdenitrifyingconditionsinsediments,we hereby report on the ability of the bacterium Pseudo-monassp. strain ADP to rapidly mineralize atrazine inaquifer sediments under nitrate reducing conditions. Whenatrazine was present in low concentrations (relevant tononpoint sources such as agricultural application),the bacterium mineralized 55% and 75% of the atrazine in2 and 4 days, respectively. When atrazine was presentin high concentrations (relevant to spill sites), P. ADPmineralized 48% and 78% in 4 and 15 days, respectively.The present study indicates that bioaugmantation with aneffective atrazine mineralizing bacterium such as P. ADPcouldyieldhighmineralizationratesevenunderoxygenlimitedconditions and have a significant implication for biore-mediation of atrazine in contaminated aquifers.

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance 1 H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater.

Atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine, is metabolized relatively slowly in natural soils and waters by resident microorganisms. Recently, several atrazine-degrading bacterial pure cultures were isolated and the molecular basis of bacterial atrazine metabolism is now beginning to be revealed. Pseudomonas sp. strain ADP was isolated from a herbicide spill site for its ability to use atrazine as the sole source of nitrogen for growth. Atrazine metabolism also liberated the triazine ring carbon atoms as carbon dioxide. Hydroxyatrazine was detected transiently in the growth medium during the course of atrazine metabolism. Previously, hydroxyatrazine was proposed to be derived solely from abiotic hydrolysis catalyzed by soil organic matter and clays. The gene encoding the enzymatic hydrolysis of atrazine by Pseudomonas sp. ADP was cloned and expressed in Escherichia coli. Cell-free atrazine hydrolysis activity in the recombinant E. coli strain was determined by high pressure liquid chromatography. The enzyme, atrazine chlorohydrolase, was purified to homogeneity using ammonium sulfate precipitation and ion exchange chromatography. The purified chlorohydrolase showed a single band on denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a subunit molecular weight of 60,000. Gene sequencing data yielded a molecular weight of 52,421. Gel filtration chromatography indicated a holoenzyme molecular weight of 240,000 consistent with an α4 or α5 subunit stoichometry. In [18O]-H2O, atrazine chlorohydrolase yielded [18O]-hydroxyatrazine quantitatively. In control experiments incubated and analyzed under the same conditions, [18O] from H2O did not exchange into hydroxyatrazine. These data are consistent with enzymatic hydrolysis of atrazine. Other bacteria were also demonstrated to catalyze atrazine hydrolysis, suggesting this biologically-mediated reaction is widespread in soil and water.

Atrazine chlorohydrolase was purified from cell extracts of Pseudomonas sp. strain ADP and immobilized in sol–gel matrices of different hydrophobicity. Experiments were made with various amounts of water and under different drying conditions. Under optimal conditions, more than 40% of the activity of the free enzyme was maintained in the sol–gel matrix for more than 3 weeks. The more hydrophobic sol–gel matrices gave the best results in maintaining atrazine chlorohydrolase activity.

Pythium damping-off and root-rot are among the most important soilborne diseases of greenhouse plants and seedlings grown in container media. It has been shown previously that composts may be conducive, suppressive or partially suppressive to Pythium diseases. The major goal of this work was to investigate rapid, practical and reliable methods for determining of the degree of suppressiveness of container media to Pythium damping-off. Several inoculation methods were tested in greenhouse bioassays. survival of propagules in suppressive versus conducive media was studied under laboratory conditions. Although both greenhouse and laboratory tests could indicate disease suppression, a bioassay with cucumber seedlings is suggested to be the most simple, effective and comprehensive method for testing suppression of Pythium diseases in compost amended container media.

Irrigation with treated waste water (TWW) for an extended period of time modified the conduciveness of soils to microbial degradation of atrazine (2-chloro,4-ethylamino,6-isopropylamino,s-triazine). Only one of the tested soils exhibited intrinsic ability to mineralize atrazine within a period of 35 days of incubation (Tzora soil, not irrigated with TWW). In the corresponding soil irrigated with TWW no atrazine mineralization could be detected, In soils augmented with Pseudomonas sp. strain ADP (an atrazine-degrading bacterium), the atrazine mineralization pattern depended on TWW irrigation history, and on pre-incubation of atrazine in the soil before inoculation with Pse. ADP. In soils irrigated with TWW and inoculated with Pse. ADP one day after atrazine application, rapid mineralization occurred and more than 80% of the atrazine was mineralized in 10 days in all soils (inoculated with 10(6) cells g(-) dry soil). However, in the corresponding soils nor irrigated with TWW, a longer lag phase was observed and only 40-60% mineralization was recorded after equivalent incubation times. A different degradation pattern was observed when atrazine was pre-incubated for 12 days in the soils before inoculation with Pse. ADP. In soils irrigated with TWW, atrazine mineralization was retarded and less than 20% of it was mineralized within 20 days, while in the corresponding soils not irrigated with TWW, atrazine mineralization reached 60-80%. From our results it is evident that aging of the herbicide in the soil has a pronounced effect on the mineralization results. Adsorption of atrazine was positively correlated with organic matter content in the TWW-irrigated soils and may be partially responsible for the inhibition of mineralization after long pre-incubation of atrazine in soil. This observation suggests that if atrazine-degrading bacteria are present in the soil, long-term TWW irrigation could enhance mineralization of newly applied atrazine in the first days after application, but would adversely affect mineralization of atrazine which had been aged in the soil for several days. Since the atrazine half-life in soils is in the order of weeks, it is to be expected that the overall effect of TWW irrigation on atrazine degradation would be toward retardation of mineralization rates. (C) 1997 Elsevier Science B.V.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.

The extent to which aquifer microbiota can be studied under laboratory or simulated conditions is limited by our inability to authentically duplicate natural conditions in the laboratory. Therefore, extrapolation of laboratory results to real aquifer situations is often criticized, unless validation of the data is performed in situ. Reliable data acquisition is critical for the estimation of chemical and biological reaction rates of biodegradation processes in groundwater and as input data for mathematical models. Typically, in situ geobiochemical studies relied on the injection of groundwater spiked with compounds or bacteria of interest into the aquifer, followed by monitoring the changes over time and space. In situ microcosms provide a more confined study site for measurements of microbial reactions, yet closer to natural conditions than laboratory microcosms. Two basic types of in situ aquifer microcosm have been described in recent years, and both originated from in situ instruments initially designed for geochemical measurements. Gillham et al. [Ground Water 28 (1990) 858–862] constructed an instrument that isolates a portion of an aquifer for in situ biochemical rate measurements. More recently Shati et al. [Environ. Sci. Technol. 30 (1996) 2646–2653] modified a multilayer sampler for studying the activity of inoculated bacteria in a contaminated aquifer. Keeping in mind recent advances in environmental microbiology methodologies such as immunofluorescence direct counts, oligonucleotide and PCR probes, fatty acid methyl esther analysis for the detection and characterization of bacterial communities, measurement of mRNA and expression of proteins, it is evident that much new information can now be gained from in situ work. Using in situ microcosms to study bioremediation efficiencies, the fate of introduced microorganisms and general geobiochemical aquifer processes can shed more realistic light on the microbial underworld. The aim of this paper is to emphasize the importance of in situ studies and to describe two different concepts of construction and application of in situ microcosms for studying microbial activity in aquifers. The overall goal is to promote the development and utilization of these valuable and largely unexplored tools.

Many toxic organic compounds that have entered the environment as a result of man’s activities are biodegradable or potentially biodegradable to less toxic organic compounds or to minerals. Pesticides represent a major group of such synthetic organic compounds, with over 45 000 registered pesticides totalling 550 million kg and 6.5 billion dollars annually in the United States alone (Ritter, 1990). Among the pesticides, herbicides are the largest group, comprising some 70

Significant atrazine degradation (50%) but only 1% mineralization was detected in samples taken from the upper soil layer (0-25 cm), but not in samples taken from deeper horizons. Thin layer chromatography analysis of noninoculated soil indicated dealkylation to be a major degradation pathway with deethylatrazine favoring deisopropylatrazine. Inoculation with Pseudomonas sp. strain ADP (P.ADP) resulted in 90-100% mineralization of [C-14]atrazine in all samples after 15 days. Atrazine was degraded in the soil via dechlorination as the first mineralization step. C-source competition was not responsible for differences in initial mineralization rates. Higher organic matter content in the upper soil level did not result in a sorption-related decrease in degradation rates. It is concluded that the limiting factor for atrazine mineralization in the tested soil profiles was the absence of atrazine-mineralizing microorganisms. Therefore, bioaugmentation may be preferable to enhancement of intrinsic atrazine-degrading activity when complete atrazine mineralization is the goal of bioremediation activity.