Isolation of Cultivable Bacteria Associated with the Root of Typha latifolia in a Constructed Wetland for the Removal of Diclofenac or Naproxen

Abstract

In this work, we designed at the level of microscale, subsurface flow wetlands planted with Typha latifolia, and used to remove diclofenac and naproxen from solutions that contained them. The wetlands were operated for 105 days with a hydraulic residence time (TRH) of 5 days. In these conditions, the removal efficiencies for diclofenac and naproxen were 98.4% and 97.7%, respectively. Moreover, we identified cultivable bacteria associated with the roots of plants exposed to either diclofenac or naproxen. We obtained 898 isolates that clustered in 9 morphotypes from the roots of Typha latifolia exposed to diclofenac, and 563 isolates grouped in 7 morphotypes, in the case of naproxen. All isolates were identified by 16S ribosomal sequencing. The BLASTn analysis indicated that 16 morphotypes showed an identity higher than 95% with the 16S rDNA gene of bacteria belonging to the genus Pseudomonas. Biochemical characterization based on plant growth-promoting activities suggests that bacteria contribute to plant growth in the wetland conditions. The results indicate that Typha latifolia and bacteria associated with their roots removed diclofenac and naproxen in subsurface flow wetlands.

Efficiency of Cattail to Remove a Mixture of Pharmaceuticals in a Constructed Wetland

“…The results showed that the wetland system planted with T. latifolia removed 82.0 % of diclofenac and 74.5 % of naproxen from the mixture. These results contrast with our previous work, where the removal efficiencies were 98.4 % for diclofenac and 97.7 % for naproxen in HSSF constructed wetlands planted with Typha latifolia and packed with gravel as support materials for plants; plants in the wetlands were exposed to single solutions of diclofenac (3 mg/L) or naproxen (5 mg/L) and the HRT was 5 days [17]. According to the results, diclofenac and naproxen were detected in the effluent, indicating that these substances were not completely removed as shown in Fig.…”

“…Typha latifolia plants of 30 cm tall were planted in the HSSF-CW units and placed at intervals of 20 to 22 cm to allow the root development; the plants were grown for 90 days to achieve a vegetation coverage before experimentation according to that indicated by the U.S. Environmental Protection Agency [15]. During this period, the wetlands received a nutrient solution (1 mM NH4 + + 1.5 mM K + + 0.125 mM PO4 3− + 0.25 mM Ca 2+ ); in previous studies, this combination of nutrients favored the plant propagation and growth in greenhouse conditions, and in presence of diclofenac and naproxen, the plants did not show effects of damage such as chlorosis [16,17]. Two HSSF-CW units were planted with Typha latifolia, and the third one was unplanted.…”

“…The concentrations of diclofenac and naproxen were determined by colorimetric methods specific for each pharmaceutical. The method to determine the diclofenac concentrations is based on its reaction with iron (III) which reduces to iron (II) with potassium ferrocyanide, forming a blue-colored complex that can be measured at 710 nm; the limit of detection (LOD) was 0.12 mg/L [17,18]. The method to determine naproxen is based on the reaction between naproxen and the sulfone phthalein acid dye bromothymol blue (BTB) to form a yellow complex of ion pairs.…”

“…The plant growth during the experimentation was followed up through the determination of chlorophyll and the height of the plants [17]. The quantification of chlorophyll was done in samples of Typha leaves, by the method of Arnon [20]; nine plants were selected from the wetland exposed to the pharmaceuticals and nine plants from the control wetland, and leaves samples were taken every 15 days during the experimental time (100 days).…”

“…Typha latifolia has shown to be tolerant to diclofenac and naproxen even at high concentrations such as 5 mg/L diclofenac and 10 mg/L naproxen; tests were done in bath systems without a plant substrate [17]. In this study, the plants height and the total chlorophyll were determined for nine plants from each wetland unit (fed with diclofenac and naproxen, and in the control wetland).…”