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- Environmental factors influencing molinate biodegradation by a two-member mixed culture in rice paddy field floodwaterPublication . Barreiros, Luisa; Peres, Joana; Azevedo, Nuno F.; Manaia, Célia M.; Nunes, Olga C.Bioaugmentation is reported as a feasible approach for the treatment of pesticide contaminated environments. Aiming the development and future implementation of a bioremediation process to treat natural waters polluted with molinate, a molinate-mineralizing culture, composed of Pseudomonas chlororaphis ON1 and Gulosibacter molinativorax ON4T (designated DC1), was assayed in paddy field floodwater microcosms. The influence of abiotic (temperature, presence of other herbicides) and biotic (floodwater autochthonous microbiota) factors on molinate mineralization by culture DC1 was assessed. In parallel, the proportion of the introduced strains in filter-sterilized floodwaters was monitored by fluorescent in situ hybridization (FISH). Molinate mineralization and growth of culture DC1 were observed under all tested conditions, although the kinetic parameters (maximum specific growth and degradation rates) were significantly affected by the environmental conditions and culture media used. Additionally, these two factors were observed to have a statistically significant interaction. The lowest values of both kinetic parameters were observed at 15 °C. The presence of the herbicides propanil, bentazone and MCPA, frequently applied in rice culture protection, did not affect the degrading capacity of culture DC1. Furthermore, it was possible to infer that the autochthonous microbiota does not retard or limit molinate biodegradation, given the growth and degradation rates by culture DC1 were higher in non-sterile microcosm assays. Although G. molinativorax ON4T is known to promote the initial breakdown of molinate, P. chlororaphis ON1 appeared to be responsible to pull up the process, since higher proportions of this organism were found at the exponential growth and molinate degradation phase. Culture DC1 is efficient, versatile and presents a promising potential to be applied as a bioaugmentation tool for the remediation of environmental waters contaminated with molinate.
- Bacterial diversity and bioaugmentation in floodwater of a paddy field in the presence of the herbicide molinatePublication . Barreiros, Luisa; Manaia, Célia M.; Nunes, Olga C.This work aimed at studying variations on the diversity and composition of the bacterial community of a rice paddy field floodwater, subjected to conventional management, namely by using the herbicide molinate. The promotion of the herbicide biodegradation either by the autochthonous microbiota or by a bioaugmentation process was also assessed. This study comprehended four sampling campaigns at key dates of the farming procedures (seeding, immediately and 6 days after application of the herbicide molinate, and after synthetic fertilization) and the subsequent physic-chemical and microbiological characterization (pH, DOC and molinate contents, total cells, cultivable bacteria and DGGE profiling) of the samples. Multivariate analysis of the DGGE profiles showed temporal variations in the bacterial community structure and the Shannon's index values indicated that the bacterial diversity reached its minimum at the molinate application day. The highest bacterial diversity coincided with the periods with undetectable concentrations of the herbicide, although microcosm assays suggested that other factors than molinate may have been responsible for the decrease of the bacterial diversity. The ability of autochthonous microorganisms to degrade molinate and the influence of the herbicide on the bacterial community composition were assessed in microcosm assays using floodwater collected at the same dates. Given molinate was not degraded by autochthonous microorganisms, and considering it represents an environmental contaminant, bioaugmentation microcosms were assayed aiming the assessment of the feasibility of a bioremediation process to clean contaminated floodwater. A molinate-mineralizing culture, previously isolated, promoted molinate removal, induced alterations in the autochthonous bacterial community structure and diversity, and was undetected after 7 days of incubation, suggesting the feasibility of the process.
- Photocatalytic ozonation of urban wastewater and surface water using immobilized TiO2 with LEDs: Micropollutants, antibiotic resistance genes and estrogenic activityPublication . Moreira, Nuno F.F.; Sousa, José M.; Macedo, Gonçalo; Ribeiro, Ana R.; Barreiros, Luisa; Pedrosa, Marta; Faria, Joaquim L.; Pereira, M. Fernando R.; Castro-Silva, Sérgio; Segundo, Marcela A.; Manaia, Célia M.; Nunes, Olga C.; Silva, Adrián M.T.Photocatalytic ozonation was employed for the first time in continuous mode with TiO2-coated glass Raschig rings and light emitting diodes (LEDs) to treat urban wastewater as well as surface water collected from the supply area of a drinking water treatment plant (DWTP). Different levels of contamination and types of contaminants were considered in this work, including chemical priority substances (PSs) and contaminants of emerging concern (CECs), as well as potential human opportunistic antibiotic resistant bacteria and their genes (ARB&ARG). Photocatalytic ozonation was more effective than single ozonation (or even than TiO2 catalytic ozonation) in the degradation of typical reaction by-products (such as oxalic acid), and more effective than photocatalysis to remove the parent micropollutants determined in urban wastewater. In fact, only fluoxetine, clarithromycin, erythromycin and 17-alpha-ethinylestradiol (EE2) were detected after photocatalytic ozonation, by using solid-phase extraction (SPE) pre-concentration and LC-MS/MS analysis. In surface water, this treatment allowed the removal of all determined micropollutants to levels below the limit of detection (0.01-0.20 ng L(-1)). The efficiency of this process was then assessed based on the capacity to remove different groups of cultivable microorganisms and housekeeping (16S rRNA) and antibiotic resistance or related genes (intI1, blaTEM, qnrS, sul1). Photocatalytic ozonation was observed to efficiently remove microorganisms and ARGs. Although after storage total heterotrophic and ARB (to ciprofloxacin, gentamicin, meropenem), fungi, and the genes 16S rRNA and intI1, increased to values close to the pre-treatment levels, the ARGs (blaTEM, qnrS and sul1) were reduced to levels below/close to the quantification limit even after 3-days storage of treated surface water or wastewater. Yeast estrogen screen (YES), thiazolyl blue tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) assays were also performed before and after photocatalytic ozonation to evaluate the potential estrogenic activity, the cellular metabolic activity and the cell viability. Compounds with estrogenic effects and significant differences concerning cell viability were not observed in any case. A slight cytotoxicity was only detected for Caco-2 and hCMEC/D3 cell lines after treatment of the urban wastewater, but not for L929 fibroblasts.