RESUMO
Pristine cold oligotrophic lakes show unique physical and chemical characteristics with permanent fluctuation in temperature and carbon source availability. Incorporation of organic toxic matters to these ecosystems could alter the bacterial community composition. Our goal was to assess the effects of simazine (Sz) and 2,4 dichlorophenoxyacetic acid (2,4-D) upon the metabolic and genetic diversity of the bacterial community in sediment samples from a pristine cold oligotrophic lake. Sediment samples were collected in winter and summer season, and microcosms were prepared using a ration 1:10 (sediments:water). The microcosms were supplemented with 0.1 mM 2,4-D or 0.5 mM Sz and incubated for 20 days at 10 °C. Metabolic diversity was evaluated by using the Biolog Ecoplate™ system and genetic diversity by 16S rDNA amplification followed by denaturing gradient gel electrophoresis analysis. Total bacterial counts and live/dead ratio were determined by epifluorescence microscopy. The control microcosms showed no significant differences (P > 0.05) in both metabolic and genetic diversity between summer and winter samples. On the other hand, the addition of 2,4-D or Sz to microcosms induces statistical significant differences (P < 0.05) in metabolic and genetic diversity showing the prevalence of Actinobacteria group which are usually not detected in the sediments of these non-contaminated lacustrine systems. The obtained results suggest that contaminations of cold pristine lakes with organic toxic compounds of anthropic origin alter their homeostasis by inhibiting specific susceptible bacterial groups. The concomitant increase of usually low representative bacterial groups modifies the bacterial composition commonly found in this pristine lake.
Assuntos
Bactérias/classificação , Bactérias/metabolismo , Variação Genética , Herbicidas/metabolismo , Lagos/química , Lagos/microbiologia , Poluentes Químicos da Água/metabolismo , Ácido 2,4-Diclorofenoxiacético/metabolismo , Bactérias/genética , Carga Bacteriana , Eletroforese em Gel de Gradiente Desnaturante , Sedimentos Geológicos/microbiologia , Viabilidade Microbiana , Microscopia de Fluorescência , Técnicas de Amplificação de Ácido Nucleico , RNA Ribossômico 16S/genética , Estações do Ano , Simazina/metabolismo , TemperaturaRESUMO
The main objective of this work was to evaluate the operational stability of a laboratory-scale aerobic biobarrier designed for the treatment of water contaminated by mixtures of three herbicides frequently found in agricultural runoffs, atrazine, simazine and 2,4-dichlorophenoxyacetic acid (2,4-D). The microbial consortium used to degrade the herbicides was composed by six cultivable bacterial strains, identified as members of the genera Variovorax, Sphingopyxis, Hydrocarboniphaga, Methylobacterium, Pseudomonas and Acinetobacter. The effect caused by a seventh member of the microbial consortium, a ciliated protozoa of the genus Colpoda, on the herbicides biodegradation kinetics, was also evaluated. The biodegradation of five combinations of the herbicides 2,4-D, atrazine and simazine was studied in the biobarrier, operated in steady state continuous culture at different volumetric loading rates. In all cases, removal efficiencies determined by chemical oxygen demand (COD) and HPLC were nearly 100 %. These results, joined to the null accumulation of aromatic byproducts of atrazine and simazine catabolism, show that after 495 days of operation, in the presence of the protozoa, the adaptability of the microbial consortium to changing environmental conditions allowed the complete removal of the mixture of herbicides.
Assuntos
Reatores Biológicos/microbiologia , Herbicidas/metabolismo , Atrazina/metabolismo , Biofilmes/crescimento & desenvolvimento , Análise da Demanda Biológica de Oxigênio , Cromatografia Líquida de Alta Pressão , Simazina/metabolismoRESUMO
Simazine is an s-triazine herbicide world widely used for the control of broadleaf weeds. The influence of leaching and microorganisms on simazine attenuation in an agricultural soil long-term treated with this herbicide was studied. To elucidate the leaching potential of simazine in this soil, undisturbed soil columns amended with simazine were placed in a specially designed system and an artificial precipitation was simulated. To evaluate the simazine removal by soil microorganisms, three soil microcosm sets were established: i) control soil; ii) soil subjected to gamma irradiation (γ-soil) and iii) γ-soil inoculated with the simazine-degrading bacterium Pseudomonas sp. strain MHP41. The simazine-degrading microorganisms in soil were estimated using an indicator for respiration combined with MPN enumeration. The simazine removal in soil was monitored by GC-ECD and HPLC. In this agricultural soil the leaching of the applied simazine was negligible. The gamma irradiation decreased in more than one order of magnitude the cultivable heterotrophic bacteria and reduced the simazine-degrading microorganisms. Simazine was almost completely depleted (97%) in control soil by natural attenuation after 23 d, whereas in γ-soil only 70% of simazine was removed. The addition of the simazine-degrading strain MHP41 to γ-soil restored and upheld high stable simazine catabolic microorganisms as well as increased the simazine removal (87%). The results indicated that simazine is subjected to microbial degradation with negligible leaching in this agricultural soil and pointed out the crucial role of native microbiota in the herbicide removal.
Assuntos
Herbicidas/metabolismo , Pseudomonas/metabolismo , Simazina/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Inoculantes Agrícolas , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Raios gama , SoloRESUMO
Bioremediation is an important technology for the removal of persistent organic pollutants from the environment. Bioaugmentation with the encapsulated Pseudomonas sp. strain MHP41 of agricultural soils contaminated with the herbicide simazine was studied. The experiments were performed in microcosm trials using two soils: soil that had never been previously exposed to s-triazines (NS) and soil that had >20 years of s-triazine application (AS). The efficiency of the bioremediation process was assessed by monitoring simazine removal by HPLC. The simazine-degrading microbiota was estimated using an indicator for respiration combined with most-probable-number enumeration. The soil bacterial community structures and the effect of bioaugmentation on these communities were determined using 16S RNA gene clone libraries and FISH analysis. Bioaugmentation with MHP41 cells enhanced simazine degradation and increased the number of simazine-degrading microorganisms in the two soils. In highly contaminated NS soil, bioaugmentation with strain MHP41 was essential for simazine removal. Comparative analysis of 16S rRNA gene clone libraries from NS and AS soils revealed high bacterial diversity. Bioaugmentation with strain MHP41 promoted soil bacterial community shifts. FISH analysis revealed that bioaugmentation increased the relative abundances of two phylogenetic groups (Acidobacteria and Planctomycetes) in both soils. Although members of the Archaea were metabolically active in these soils, their relative abundance was not altered by bioaugmentation.
Assuntos
Bactérias/crescimento & desenvolvimento , Recuperação e Remediação Ambiental , Herbicidas/metabolismo , Pseudomonas/fisiologia , Simazina/metabolismo , Microbiologia do Solo , Agricultura , Bactérias/classificação , Bactérias/genética , Pseudomonas/classificação , Poluentes do Solo/metabolismo , Poluentes do Solo/toxicidade , Fatores de TempoRESUMO
s-Triazine herbicides are used extensively in South America in agriculture and forestry. In this study, a bacterium designated as strain MHP41, capable of degrading simazine and atrazine, was isolated from agricultural soil in the Quillota valley, central Chile. Strain MHP41 is able to grow in minimal medium, using simazine as the sole nitrogen source. In this medium, the bacterium exhibited a growth rate of mu=0.10 h(-1), yielding a high biomass of 4.2 x 10(8) CFU mL(-1). Resting cells of strain MHP41 degrade more than 80% of simazine within 60 min. The atzA, atzB, atzC, atzD, atzE and atzF genes encoding the enzymes of the simazine upper and lower pathways were detected in strain MHP41. The motile Gram-negative bacterium was identified as a Pseudomonas sp., based on the Biolog microplate system and comparative sequence analyses of the 16S rRNA gene. Amplified ribosomal DNA restriction analysis allowed the differentiation of strain MHP41 from Pseudomonas sp. ADP. The comparative 16S rRNA gene sequence analyses suggested that strain MHP41 is closely related to Pseudomonas nitroreducens and Pseudomonas multiresinovorans. This is the first s-triazine-degrading bacterium isolated in South America. Strain MHP41 is a potential biocatalyst for the remediation of s-triazine-contaminated environments.
Assuntos
Pseudomonas/classificação , Pseudomonas/metabolismo , Simazina/metabolismo , Microbiologia do Solo , Atrazina/metabolismo , Técnicas de Tipagem Bacteriana , Biomassa , Biotransformação , Chile , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes Bacterianos , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Filogenia , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Using a successive transfer method on mineral salt medium containing simazine, a microbial community enriched with microorganisms able to grow on simazine was obtained. Afterwards, using a continuous enrichment culture procedure, a bacterial community able to degrade simazine from an herbicide formulation was isolated from a chemostat. The continuous selector, fed with a mineral salt medium containing simazine and adjuvants present in the commercial herbicide formulation, was maintained in operation for 42 days. Following the lapse of this time, the cell count increased from 5 x 10(5) to 3 x 10(8) CFU mL(-1), and the simazine removal efficiency reached 96%. The chemostat's bacterial diversity was periodically evaluated by extracting the culture's bacterial DNA, amplifying their 16S rDNA fragments and analyzing them by thermal gradient gel electrophoresis. Finally, a stable bacterial consortium able to degrade simazine was selected. By PCR amplification, sequencing of bacterial 16S rDNA amplicons, and comparison with known sequences of 16S rDNA from the NCBI GenBank, eight bacterial strains were identified. The genera, Ochrobactrum, Mycobacterium, Cellulomonas, Arthrobacter, Microbacterium, Rhizobium and Pseudomonas have been reported as common degraders of triazinic herbicides. On the contrary, we were unable to find reports about the ability of the genus Pseudonocardia to degrade triazinic compounds. The selected bacterial community was attached to a porous support in a concurrently aerated four-stage packed-bed reactor fed with the herbicide. Highest overall simazine removal efficiencies eta (SZ) were obtained at overall dilution rates D below 0.284 h(-1). However, the multistage packed bed reactor could be operated at dilution rates as high as D = 3.58 h(-1) with overall simazine removal volumetric rates R (v,SZ) = 19.6 mg L(-1) h(-1), and overall simazine removal specific rates R (X,SZ) = 13.48 mg (mg cell protein)(-1) h(-1). Finally, the consortium's ability to degrade 2-chloro-4,6-diamino-1,3,5-triazine (CAAT), cyanuric acid and the herbicide atrazine, pure or mixed with simazine, was evaluated in fed batch processes.
Assuntos
Bactérias/classificação , Bactérias/metabolismo , Biodiversidade , Reatores Biológicos/microbiologia , Herbicidas/metabolismo , Simazina/metabolismo , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Biofilmes , Contagem de Colônia Microbiana , Meios de Cultura/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida/métodos , Desnaturação de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Temperatura , Fatores de TempoRESUMO
Characterization of pesticide bioavailability, particularly in aged soils, is of continued interest because this information is necessary for environmental risk assessment. However, pesticide bioavailability in aged soils has been characterized by a variety of methods with limited success, due in part to methodological limitations. The objective of this study was to use solvent extraction methods to correlate simazine residue bioavailability in aged soils to simazine mineralization using a simazine-mineralizing bacterium. Soils from Brazil, Hawaii, and the midwestern United States were treated with UL-ring-labeled [14C]simazine and incubated for up to 8 weeks. At the end of each incubation period, soils were either incubated further, extracted with 0.01 M CaCl2, or extracted with aqueous methanol (80:20 v/v methanol/water). In a parallel experiment, after each incubation period, soils were inoculated with the bacterium Pseudomonas sp. strain ADP, which is capable of rapidly mineralizing simazine, and 14CO2 was determined. The inoculated soil samples were then extracted with 0.01 N CaCl2 and with aqueous methanol. This allowed for the evaluation of the bioavailability of aged simazine residues, without the contribution of simazine desorption from soil. Results of these studies indicated that simazine sorption to soil increased with aging and that amounts of simazine in aged soils extracted by 0.01 M CaCl2 and aqueous methanol were highly correlated to amounts of simazine mineralized by Pseudomonas sp. strain ADP. Consequently, 0.01 M CaCl2/methanol-extractable simazine in aged soils can be used to estimate bioavailable residues. This technique may be useful in determining the bioavailability of other s-triazine compounds in soils.