Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene

Abstract

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.

FIG. 1

Dechlorination of trichlorobenzenes by a culture growing from a small inoculum with 10 mM pyruvate as the carbon and energy source in Ti(III)-reduced medium. Symbols: •, 1,2,3-trichlorobenzene; ▪, 1,2,4-trichlorobenzene; ○, 1,3-dichlorobenzene; □, 1,4-dichlorobenzene.

FIG. 2

Concentrations of metabolites, growth, and dichlorobenzene formation in cultures initially supplied with 10 mM pyruvate in Ti(III)-reduced medium containing 20 μM sulfide as a source of sulfur. Symbols: (A) ○, pyruvate; □, acetate; ▿, formate; ▵, propionate; ⊕, methane; (B) ⧫, formation of dichlorobenzenes; •, protein; ▪, hydrogen. Methane and hydrogen data are given as the nominal concentrations. Data are means of triplicate cultures; standard deviations were below 10%.

FIG. 3

Effect of specific inhibitors on the dechlorination of trichlorobenzenes in Ti(III)-reduced medium containing 10 mM pyruvate. Symbols: ○, control; □, 4 mM BES; ▪, 4 mM BES plus 15% (vol/vol) hydrogen; ▿, 2 mM molybdate; ▵, negative control, not inoculated. Data are means of triplicate cultures; standard deviations were below 10%.

FIG. 4

Dechlorination of trichlorobenzenes depends on the initial sulfate concentration. All cultures were Ti(III) reduced and contained 10 mM pyruvate. Symbols: ○, dichlorobenzene formation; □, hydrogen; ▿, sulfide; ▵, sulfate. Hydrogen data are given as nominal concentrations. Data represent values 7 days after inoculation and are means of triplicate cultures; standard deviations were below 10%.

FIG. 5

Metabolic activities in a Ti(III)-reduced culture containing 10 mM pyruvate and 2 mM sulfate. Symbols: ○, pyruvate; □, protein; ▿, sulfide; ▵, formation of dichlorobenzenes. Data are means of triplicate cultures ± standard deviations.