Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing

Abstract

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.

FIG. 1.

Metabolism of phthalate isomers by enrichment cultures after more than 10 transfers. (a) Enrichment with ortho-phthalate from sludge I, enrichment UP; (b) enrichment with isophthalate from sludge I, enrichment UI; (c) enrichment with terephthalate from sludge I, enrichment UT; (d) enrichment with ortho-phthalate from sludge II, enrichment JP; (e) enrichment with isophthalate from sludge II, enrichment JI; (f) enrichment with terephthalate from sludge II, enrichment JT.

FIG. 2.

Phylogenetic trees among the group ‘Desulfotomaculum lineage I' (A) and the class delta-Proteobacteria (B) based on comparative analyses of 16S rRNA gene sequences, showing the phylogenetic positions of clones and an isolated bacterium (strain JT) obtained in this study (clones UP, UI, and UT were retrieved from enrichment cultures with sludge I as the inoculum, and clones JP, JI, and JT were obtained from enrichments with sludge II as the inoculum). The trees were calculated based on a distance matrix analysis of 16S rRNA gene sequences (neighbor-joining tree). The scale bars represent the number of nucleotide changes per sequence position. The symbols at nodes correlate with the bootstrap values (percent) obtained with 1,000 resamplings.

FIG. 3.

In situ hybridization of phthalate isomer-degrading enrichment cultures. The enrichment cultures were simultaneously hybridized with indodicarbocyanine-labeled Bacteria-specific probe EUB338 (green, panel 3) and indocarbocyanine-labeled clone-specific probes (red, panel 2). Phase-contrast micrographs (panel 1) and fluorescence micrographs of the same fields (panels 2 and 3) are shown. (A) JT culture (terephthalate culture with sludge II) hybridized with probe JPIT74, specific for clones JP, JI, and JT in subcluster Ih of ‘Desulfotomaculum lineage I'; (B) UT culture (terephthalate enrichment with sludge I) hybridized with probe UT62, specific for clone UT in subcluster Ih of ‘Desulfotomaculum lineage I'; (C) UI culture (isophthalate enrichment with sludge I) hybridized with the UI178 probe, specific for clone UI in the delta-Proteobacteria. Bars, 10 μm. Arrows indicate methanogen-like cells.

FIG. 4.

Phase-contrast micrographs showing the cell morphology of strain JT grown on crotonate (10 mM) (A) and on terephthalate (1 mM) in coculture with Methanospirillum hungatei (B). Bars, 10 μm.

FIG. 5.

Terephthalate degradation by strain JT in coculture with Methanospirillum hungatei.