Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen

Abstract

Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.

FIG. 1.

Phase-contrast micrographs of strain UI^T (indicated by white arrows) in coculture with M. hungatei, grown on 2 mM isophthalate (A) and grown on 2 mM phenol (B). Bars, 10 μm.

FIG. 2.

Thin-section electron micrographs of strain UI^T in coculture with M. hungatei grown on 4-hydroxybenzoate (3 mM), illustrating typical gram-negative cell wall structure (OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane). The bar for the large panel represents 0.5 μm. The inset of the panel shows a magnified view of the cell wall structure (bar, 0.25 μm).

FIG. 3.

Isophthalate (a), phenol (b), and 4-hydroxybenzoate (c) degradation by strain UI^T in coculture with M. hungatei. The concentrations are shown in millimoles liter-culture⁻¹ (left) and in millimolar (right).

FIG. 4.

Phylogenetic position of strain UI^T among members of the class Deltaproteobacteria. Group TA (Syntrophorhabdaceae) previously consisted only of environmental 16S rRNA gene clone sequences that were mainly retrieved from anaerobic sludge treating terephthalate-containing wastewater. The tree was calculated on the basis of a distance matrix analysis of 16S rRNA gene sequences (neighbor-joining tree). The scale bar represents the number of changes in nucleotide per sequence position. The numbers at the nodes show the bootstrap values (as a percentage) obtained with 1,000 resampling analysis. The GenBank accession numbers are shown after the clone. TCB, trichlorobenzene; UASB, upflow anaerobic sludge blanket.

FIG. 5.

Models of syntrophic phenol degradation and isophthalate degradation by strain UI^T with different partners and intermediate compounds formed in the processes. The electron shuttle for interspecies electron transfer was assumed to be a proton in the models, since an increase in hydrogen partial pressures (ca. 20 Pa) was observed in all degradation processes. However, it may be possible that other external electron carriers can also intermediate in the syntrophic reactions. Intermediate compounds detected experimentally and possible degradation processes of these compounds were also shown in the models.