Anaerobe isolation from denitrifying benzene-degrading enrichment culture and their capacity to mineralize benzene

Only a few benzene-mineralizing anaerobes have been isolated to date. In an attempt using classical isolation techniques to isolate benzene-mineralizing pure cultures from a benzene-mineralizing nitrate-reducing microbial community, two putative isolates were gained under nitrate-reducing conditions spiked separately with acetate and benzene as sole sources of carbon and energy with media containing ammonium or without ammonium. Both putative isolates; Bz4 (with ammonium) and Bz7 (without ammonium) - mineralized 13C-labelled acetate under anoxic conditions at 3.3 and 2.7 M day-1, respectively, revealed by analysis of evolved 13CO2. However, only Bz4 mineralized [13C6]-labelled benzene (0.298 M benzene mineralized day-1) generated up to 960.2 {+/-} 0.3 {per thousand} {delta}13C-CO2 during 184 days while producing only slight amounts of nitrite (4.60 {+/-} 0.004 M); no benzene was mineralized by Bz7 during 184 d, and no nitrite was detected. The 16S rRNA gene amplicon sequencing of the acetate-grown bacteria revealed consortia enriched in Nocardioides (8.9%), Pseudomonas (18.2%), Rhizobiaceae (21.0%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (51.4%) for Bz4 and Simplicispira (96.7%) for Bz7. The gained Bz4 consortium that mineralized benzene under anoxic condition can be further purified and explored for their metabolic potentials.