Exploring benzene mineralization by anaerobes isolated from denitrifying enrichment cultures

Abstract

Objectives

The study aimed to isolate and characterize benzene-mineralizing anaerobes from a nitrate-reducing community. The goal was to evaluate their potential for benzene degradation under anoxic conditions and to compare composition and metabolic activity in media with and without ammonium.

Methods

Two putative isolates (Bz4 and Bz7) were obtained using classical isolation techniques under nitrate-reducing conditions with either acetate or benzene as the sole carbon and energy source. Enrichment media differed by the presence (Bz4) or absence (Bz7) of ammonium. The ability of the isolates to mineralize [¹³C]-labeled acetate and [¹³C₆]-labeled benzene was assessed by monitoring ¹³CO₂ production under anoxic conditions over 184 days. Nitrite production was also measured. Community composition was determined via 16S rRNA gene amplicon sequencing.

Results

Both Bz4 and Bz7 consortia mineralized [¹³C]-acetate anoxically, with mineralization rates of 3.3 μM day⁻¹ and 2.7 μM day⁻¹, respectively. Benzene mineralization was observed only in Bz4, which degraded [¹³C₆]-benzene at a rate of 0.298 μM day⁻¹, resulting in ¹³CO₂ production with δ¹³C values reaching 960.2 ± 0.3 ‰. Bz4 also produced nitrite (4.60 ± 0.004 μM), while Bz7 showed no benzene mineralization with a smaller quantity of nitrite production. Microbial community analysis revealed that Bz4 was dominated by Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (51.4 %), Rhizobiaceae (21.0 %), Pseudomonas (18.2 %), and Nocardioides (8.9 %). In contrast, Bz7 was almost exclusively composed of Simplicispira (96.7 %).

Conclusions

The Bz4 consortium demonstrated effective anaerobic benzene mineralization under nitrate-reducing conditions, highlighting its potential for further purification and study in the context of anaerobic benzene biodegradation.