Characterization of pure cultures isolated from sulfamethoxazole-acclimated activated sludge with respect to taxonomic identification and sulfamethoxazole biodegradation potential

Abstract

Background: Sulfamethoxazole (SMX, sulfonamide antibiotic) biodegradation by activated sludge communities (ASC) is still only partly understood. The present work is focusing on nine different bacteria species capable of SMX biodegradation that were isolated from SMX-acclimated ASC.

Results: Initially 110 pure cultures, isolated from activated sludge, were screened by UV-absorbance measurements (UV-AM) for their SMX biodegradation potential. Identification via almost complete 16S rRNA gene sequencing revealed five Pseudomonas spp., one Brevundimonas sp., one Variovorax sp. and two Microbacterium spp.. Thus seven species belonged to the phylum Proteobacteria and two to Actinobacteria. These cultures were subsequently incubated in media containing 10 mg L(-1) SMX and different concentrations of carbon (sodium-acetate) and nitrogen (ammonium-nitrate). Different biodegradation patterns were revealed with respect to media composition and bacterial species. Biodegradation, validated by LC-UV measurements to verify UV-AM, occurred very fast with 2.5 mg L(-1) d(-1) SMX being biodegraded in all pure cultures in, for UV-AM modified, R2A-UV medium under aerobic conditions and room temperature. However, reduced and different biodegradation rates were observed for setups with SMX provided as co-substrate together with a carbon/nitrogen source at a ratio of DOC:N - 33:1 with rates ranging from 1.25 to 2.5 mg L(-1) d(-1).

Conclusions: Media containing only SMX as carbon and nitrogen source proved the organisms' ability to use SMX as sole nutrient source where biodegradation rates decreased to 1.0 - 1.7 mg L(-1) d-(1). The different taxonomically identified species showed specific biodegradation rates and behaviours at various nutrient conditions. Readily degradable energy sources seem to be crucial for efficient SMX biodegradation.

Figure 1

Maximum likelihood-based trees reflecting the phylogeny and diversity of the isolated nine species capable of SMX biodegradation based on nearly complete 16S rRNA gene sequence comparisons. Phylogenetic tree calculated for A)Pseudomonas spp., Variovorax spp. and Brevundimonas spp. and B) for Microbacterium spp.. The tree shows the sequences obtained in this study (bold text) and their next published relatives according to the NCBI database (plain text). Numbers preceding taxonomic names represent EMBL sequence accession numbers. Scale bar indicates 0.01% estimated sequence divergence.

Figure 2

Aerobic SMX biodegradation patterns of pure cultures in MSM-CN media. A, B) measured with UV-AM, initial SMX concentration 10 mg L^-1. C, D) LC-UV analyses of SMX concentrations in the used pure cultures in MSM-CN. Determination was performed at experimental startup, after 4 and 10 days to verify UV-AM values. Asterisks indicate measured values below limit of detection. Shown are mean values of SMX absorbance in duplicate experiments. Standard deviations were too low to be shown (<1%).

Figure 3

Aerobic SMX biodegradation patterns of pure cultures in MSM media. A, B) measured with UV-AM, initial SMX concentration 10 mg L^-1. C, D) LC-UV analyses of SMX concentrations in the pure cultures in MSM at experimental startup, after 4 and 10 days to validate UV-AM. Asterisks indicate measured values below limit of detection. Shown are mean values of SMX absorbance in duplicate experiments. Standard deviations were too low to be shown (<1%).

Figure 4

Aerobic SMX biodegradation patterns of pure cultures in R2A-UV media. A) measured with UV-AM, initial SMX concentration 10 mg L^-1. B) LC-UV analyses of SMX concentrations within the nine pure cultures in R2A-UV media performed at experimental startup, after 4 and 10 days to verify the results of UV-AM. Asterisks indicate measured values below limit of detection. Shown are mean SMX absorbance values of duplicate experiments. Standard deviations were too low to be shown (<1%).