Physicochemical characterization of Pseudomonas stutzeri UFV5 and analysis of its transcriptome under heterotrophic nitrification/aerobic denitrification pathway induction condition

Abstract

Biological ammonium removal via heterotrophic nitrification/aerobic denitrification (HN/AD) presents several advantages in relation to conventional removal processes, but little is known about the microorganisms and metabolic pathways involved in this process. In this study, Pseudomonas stutzeri UFV5 was isolated from an activated sludge sample from oil wastewater treatment station and its ammonium removal via HN/AD was investigated by physicochemical and molecular approaches to better understand this process and optimize the biological ammonium removal in wastewater treatment plants. Results showed that P. stutzeri UFV5 removed all the ammonium in 48-72 hours using pyruvate, acetate, citrate or sodium succinate as carbon sources, C/N ratios 6, 8, 10 and 12, 3-6% salinities, pH 7-9 and temperatures of 20-40 °C. Comparative genomics and PCR revealed that genes encoding the enzymes involved in anaerobic denitrification process are present in P. stutzeri genome, but no gene that encodes enzymes involved in autotrophic nitrification was found. Furthermore, transcriptomics showed that none of the known enzymes of autotrophic nitrification and anaerobic denitrification had their expression differentiated and an upregulation of the biosynthesis machinery and protein translation was observed, besides several genes with unknown function, indicating a non-conventional mechanism involved in HN/AD process.

Figure 1

Phylogenetic tree based on the partial sequence of the 16S rRNA of the UFV5 isolate, reference sequence (T) and sequences of HN/AD microorganisms belonging to the same genus described in the literature. The tree was constructed by the neighbor joining method with a bootstrap value represented in the branches referring to 1000 replications. The accession numbers of the GenBank sequences are shown in parentheses. The Nitrososphaera viennensis sequence was added as the outgroup.

Figure 2

Percentage of ammonium removal from the P. stutzeri UFV5 isolate over time. Cultures were incubated in HNM medium for seven days, and ammonium quantification was monitored every 24 hours. Activated sludge was used as a positive control.

Figure 3

Production of gaseous nitrogen by P. stutzeri UFV5 bacteria during the ammonium removal under aerobic conditions. The positive control of the technique was performed by the injection of pure atmospheric air, the positive control of the biological process was HNM medium inoculated with nitrifying activated sludge, and negative control was HNM medium without inoculum. Axis X = time, in minutes, of gas detection, and axis Y = signal intensity, the larger the peak, the greater the amount of gaseous nitrogen produced.

Figure 4

Effect of physical-chemical factors on growth () and ammonium removal (■) of bacterial isolate P. stutzeri UFV5 after 72 hours of incubation in HNM medium. (a) Carbon source - the carbon sources used were sodium pyruvate (SP), sucrose (S), sodium acetate (SA), sodium citrate (SC), sodium succinate (SS) and glucose (G)), (b) pH - 3, 5, 7 and 9, (c) Carbon/Nitrogen Ratio - 4, 6, 8, 10 and 12, (d) Salinity - 0, 3, 6, 9, 12 and 15% of NaCl and (e) Temperature - 20, 25, 30, 35 and 40 °C. The graph represents the mean values of ammonium removal and optical density and their respective standard deviations. Ammonium removal means followed by at least one same letter did not differ at a 5% level of significance as determined by Tukey’s test.

Figure 5

Ammonium consumption by optical density unit (normalized values) of P. stutzeri UFV5 at different concentrations of ammonium. Analyses were performed every 2 hours over the 10-hour period.

Figure 6

Transcriptome analysis of the isolate P. stutzeri UFV5 in low and high concentrations of ammonium. (A) Venn diagram showing the number of genes that were expressed specifically in each condition and those expressed in both, and in the table below the figure are described the genes that were expressed with statistical significance exclusively in each condition; (B) Volcano plot to show differentially expressed genes regulated negatively and positively in both treatments: red = negatively regulated expression genes with statistical significance; green = positively regulated expression genes with statistical significance; blue = expression genes unaltered, and in the table below the figure are the annotation with fold change of those genes represented by the red and green dots.

Figure 7

Differentially expressed genes under conditions of low and high ammonium concentrations that were up regulated. (a) Network of interactions of the genes based on their functions obtained by the STRING program; (b) Enrichment analysis of biological processes with increasing ammonium concentration by the GOanna software. The colored balls indicate the enriched processes - the stronger the yellow, the more enriched the process was, and the white balls indicate unenriched processes that serve as connectors to the network.