IN-SITU ANALYSIS OF DENITRIFYING TOLUENE-DEGRADING AND M-XYLENE-DEGRADING BACTERIA IN A DIESEL FUEL-CONTAMINATED LABORATORY AQUIFER COLUMN

A diesel fuel-contaminated aquifer was bioremediated in situ by the in jection of oxidants (O-2 and NO3-) and nutrients in order to stimulate microbial activity. After 3.5 years of remediation, an aquifer sample was excavated and the material was used (i) to isolate bacterial stra ins able to grow on selected hydrocarbons under denitrifying condition s and (ii) to construct a laboratory aquifer column in order to simula te the aerobic and denitrifying remediation processes. Five bacterial strains isolated from the aquifer sample were able to grow on toluene (strains T-2 to T-4, T-6, and T-10), and nine bacterial strains grew o n toluene and m-xylene (strains M-3 to M-7 and M-9 to M-12). Strains T -2 to T-4, T-6, and T-10 were cocci, and strains M-3 to M-7 and M-9 to M-12 were rods. The morphological and physiological differences were also reflected in small sequence variabilities in domain III of the 23 S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S r RNA of one isolate (T-3 and M-3) of each group revealed a close phylog enetic relationship for both groups of isolates to organisms of the ge nus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcu s tolulyticus group, and Azoarcus evansii were used to investigate the significance of hydrocarbon-degrading Azoarcus spp. in the laboratory aquifer column. The number of bacteria in the column determined after DAPI (4',6-diamidino-2-phenylindole) staining was 5.8 x 10(8) to 1.1 x 10(9) cells g of aquifer material(-1). About 1% (in the anaerobic zo ne of the column) to 2% (in the aerobic zone of the column) of these b acteria were detectable by using a combination of probes Azo644 and Az o1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are sign ificant members of the indigenous microbiota. More than 90% of the tot al number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the beta subdivision of the class Proteobacteria (beta-Proteobacteria) , and 10 to 16% belonged to the gamma-Proteobacteria. Bacteria of the alpha-Proteobacteria were present in high numbers (10%) only in the ae robic zone of the column.