Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge

Abstract

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages--GordTnk2, Gmala1, and GordDuk1--had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5-15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment.

Figure 1. Phylogenetic tree derived from 16S rDNA gene sequences, created using the neighbour-joining method with gap positions excluded.

Numbers on the tree indicate bootstrap percentages (from 1000 replicates) for branch points. Assignments of identities of the Gordonia isolates are indicated. Strains G5 and G11 were highly similar to G. sputi, G. aichiensis and G. otitidis and thus were identified to the genus level only.

Figure 2. Transmission electron micrographs of phages (A) GordTnk2, (B) Gmala1, (C) GordDuk1, and (D) Gsput1.

Samples were stained with 2% (w/v) uranyl acetate and observed at 100 kV. Scale bar is 100 nm.

Figure 3. Genome map of Gordonia phages GordTnk2 and Gsput1.

GordTnk2 is presented to represent Gmala1 and GordDuk1, as these three phages share great similarity in genome arrangement. Predicted genes are represented by boxes above and below the heavy black line; boxes above the lines are genes encoded on the forward strand, and those below the lines are on the reverse strand. The ruler below the genomes indicates the scale (in kb). Genome features are color coded according to the legend. (A) Comparison map of GordTnk2 to Gordonia phage GTE7 (accession No. NC_016166), and Rhodococcus equi phage ReqiDocB7 (accession No. NC_023706). Proteins sharing identities (e value < 10⁻⁵) among three phages were identified and color coded according to the legend. (B) Genome map of Gsput1. Proteins sharing identities (e value < 10⁻⁵) with Mycobacterium phage were identified and the clusters or singletons (Sgn) of the Mycobacterium phage are indicated in red letters. Abbreviations: TerS, terminase small subunit; TerL, terminase large subunit; tmp, tape measure protein; Prim, primase; Pol, DNA polymerase; DNA MTase, DNA methylase; HNH, homing endonuclease; Int, integrase; vWA, Von Willebrand factor type A domain protein; AAA, ATP-hydrolyzing domain protein; RecA, recombinase; Nuc, nuclease.

Figure 4. Lysis genes in Gordonia phages GordTnk2, Gmala1, and GordDuk1, and their comparisons to Gordonia phage GTE7 and Rhodococcus equi phage ReqiDocB7.

Predicted genes are represented by boxes above and below the black line (encoded on the forward and reverse strand, respectively). The ruler below the genes indicates the scale (in kb). Genome features are color coded according to the legend. Lysis and holin proteins sharing identities (e value < 10⁻⁵) among presented phages were linked with blue lines. The number of predicted transmembrane domains in holin is indicated above the protein.

Figure 5. Effect of phage on total bacterial and Gordonia levels in laboratory-scale activated sludge systems.

Gordonia strains G11 and G7 were inoculated to activated sludge with (empty mark) or without (filled mark) phage cocktail treatment. Quantities (expressed as genome equivalents, or GEq per ml sludge) of total bacterial (A), Gordonia G11 (B), Gordonia G7 (C), percentage of total bacteria of Gordonia G11 (D) and G7 (E) were determined. Of each treatment (with or without phage), the mean values of two parallel sludge systems were plotted, with the error bars indicating the standard deviations.

Figure 6. Recoverability of phage against Gordonia in the laboratory-scale activated sludge systems.

Gordonia strains G11 and G7 were inoculated to activated sludge with (empty mark) or without (filled mark) phage cocktail treatment. Mean levels of phage against host G11 (A), and host G7 (B), in two parallel sludge systems were plotted with error bars indicating standard deviations.