Removal of Ibuprofen in Water by Bioaugmentation with Labrys neptuniae CSW11 Isolated from Sewage Sludge-Assessment of Biodegradation Pathway Based on Metabolite Formation and Genomic Analysis

Abstract

Ibuprofen (IBP) is one of the most consumed drugs in the world. It is only partially removed in wastewater treatment plants (WWTPs), being present in effluent wastewater and sewage sludge, causing the widespread introduction of IBP as an emergent xenobiotic in different environmental compartments. This study describes the use of Labrys neptuniae CSW11, recently described as an IBP degrader, through bioaugmentation processes for the removal of IBP from water under different conditions (additional carbon sources, various concentrations of glucose and IBP). L. neptuniae CSW11 showed very good results in a wide range of IBP concentrations, with 100% removal in only 4 days for 1 and 5 mg L^-1 IBP and 7 days for 10 mg L^-1, and up to 48.4% removal in 28 days for IBP 100 mg L^-1 when using glucose 3 g L^-1 as an additional carbon source. Three IBP metabolites were identified during the biotransformation process: 1-hydroxyibuprofen (1-OH-IBP), 2-hydroxyibuprofen (2-OH-IBP), and carboxyibuprofen (CBX-IBP), whose concentrations declined drastically in the presence of glucose. IBP metabolites maintained a certain degree of toxicity in solution, even when IBP was completely removed. The results indicate that L. neptuniae CSW11 can be quite effective in degrading IBP in water, but the bioaugmentation method should be improved using CSW11 in consortia with other bacterial strains able to degrade the toxic metabolites produced. A genome-based analysis of L. neptuniae CSW11 revealed different enzymes that could be involved in IBP biodegradation, and a potential metabolic pathway was proposed based on the metabolites observed and genome analysis.

Keywords: Labrys neptuniae; bacterial remediation; ibuprofen; metabolites; toxicity; wastewater.

Figure 1

Ibuprofen biotransformation capability by Labrys neptuniae CSW11 in the presence of acetate, yeast extract or glucose (1 g L⁻¹) as additional carbon sources, in comparison to control experiments without inoculation with CSW11. Error bars indicate standard deviation.

Figure 2

Ibuprofen (10 mg L⁻¹) biotransformation capability by Labrys neptuniae CSW11 in the absence and in the presence of glucose (1 and 3 g L⁻¹) in comparison to control experiments without inoculation with CSW11. Error bars indicate standard deviation.

Figure 3

Influence of different ibuprofen concentrations on the removal efficiency of Labrys neptuniae CSW11 in the presence of glucose 1g L⁻¹ (A) and 3g L⁻¹ (B). Error bars indicate standard deviation.

Figure 4

Maximum likelihood tree based on 16S rRNA genes showing the phylogenetic relationships between the strain L. neptuniae CSW11 isolated from the ibuprofen enrichment culture and other species of the same genus. Rhizorhabdus wittichii MPO218 was used as an outgroup. Bootstrap values shown at the branch nodes are based on 1000 replicates. The scale bar represents 0.02 substitution per nucleotide. GenBank accession numbers are in parentheses.

Figure 5

Evolution of ibuprofen (10 mg L⁻¹) and metabolites detected during its biotransformation by Labrys neptuniae CSW11 in solution in the absence (A) and presence of glucose (3 mg L⁻¹) (B).

Figure 6

Potential biodegradation pathway of ibuprofen based on metabolites detected in the culture medium of L. neptuniae CSW11 during biodegradation assays (underlined) and catabolic enzymes described by Marchlewicz et al. * [16], Salgado et al. ** [29], Diaz et al. *** [73] and Li et al. **** [7], and identified in the genome annotation of the bacterial strain CSW11. Hpd (4-hydroxyphenylpyruvate dioxygenase), HmgA (homogentisate 1,2-dioxygenase), HpaBC (4-hydroxyphenyl acetate monooxygenase), HpaD (3,4 dihydroxyphenylacetate 2,3-dioxygenase), HpaE (5-carboxymethyl-2-hydroxymuconato semialdehyde dehydrogenase), HpaF (5-carboxymethyl-2-hydroxymuconato isomerase), HpaG (5-oxo-pent-3-ene-1,2,5-tricarboxylic acid), HpaH (2-oxo-hepta-3-ene-1,7-dioic acid hydratase), HpaI (2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase), Sad (succinic semialdehyde dehydrogenase).