In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column

Abstract

A diesel fuel-contaminated aquifer was bioremediated in situ by the injection of oxidants (O 2 and NO 3 ؊) 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 strains able to grow on selected hydrocarbons under denitrifying conditions and (ii) to construct a laboratory aquifer column in order to simulate 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 on 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 23S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S rRNA of one isolate (T 3 and M 3) of each group revealed a close phylogenetic relationship for both groups of isolates to organisms of the genus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcus 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 ؋ 10 8 to 1.1 ؋ 10 9 cells g of aquifer material ؊1. About 1% (in the anaerobic zone of the column) to 2% (in the aerobic zone of the column) of these bacteria were detectable by using a combination of probes Azo644 and Azo1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are significant members of the indigenous microbiota. More than 90% of the total number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the ␤ subdivision of the class Proteobacteria (␤-Proteobacteria), and 10 to 16% belonged to the ␥-Proteobacteria. Bacteria of the ␣-Proteobacteria were present in high numbers (10%) only in the aerobic zone of the column. Diesel fuel-contaminated soils and aquifers can be partially remediated by pumping hydrocarbons occurring in free phase back to the soil surface or by stripping the subsurface with air (7). Residual hydrocarbons, however, are often trapped in cracks and pores of the subsurface, and they may be removed by in situ bioremediation. This technique is usually based on the infiltration of water supplemented with oxidants (e.g., O 2 and NO 3 Ϫ) and/or nutrients (e.g., NH 4 ϩ and PO 4 3Ϫ) to stimulate the catabolic activity of microorganisms in the subsurface and thereby the biodegradation of the hydrocarbons (18, 23-25, 32). An in situ bioremediation process was applied in a diesel fuel-contaminated aquifer in Menziken, Switzerland (23). Groundwater supplemented with O 2 (329 M) and NO 3

Anaerobic utilization of alkylbenzenes and n‐alkanes from crude oil in an enrichment culture of denitrifying bacteria affiliating with the β‐subclass of Proteobacteria

“…As all known alkylbenzene-degrading denitrifiers and even an alkane-degrading denitrifier (strain HxN1; A. Behrends, F. Rainey and F. Widdel, unpublished) belong to the Azoarcus/Thauera group (Fries et al, 1994;Anders et al, 1995;Rabus and Widdel, 1995a;Zhou et al, 1995;Hess et al, 1997), the presence of bacteria with the same affiliation was expected in the present enrichment culture. Therefore, a new probe termed AT1458 was designed that, according to the databases ARB and EMBL, targets a sequence (Escherichia coli positions 1458-1478) unique to all members of the genera Azoarcus and Thauera sequenced in this region.…”

“…As all known alkylbenzenedegrading, denitrifying bacteria belong to this group, one may conclude that Azoarcus and Thauera species are also responsible for the observed alkylbenzene utilization in the enrichment cultures. Recently, two oligonucleotide probes, Azo644 and Azo1251, were constructed according to sequences from isolated alkylbenzene-degrading denitrifiers related to Azoarcus species and used for the in situ detection of such bacteria in subsurface soil exposed to fuel hydrocarbons (Hess et al, 1997). Sequence comparison of these probes with the presently designed probe AT1458 revealed different specificities.…”

“…Therefore, anaerobic degradation of oil hydrocarbons has been most intensively studied with BTEX, in particular with toluene. Anaerobic degradation of toluene has been investigated in microbial communities (Grbić-Galić and Vogel, 1987;Kuhn et al, 1988;Edwards and Grbić-Galić, 1992) and pure cultures of denitrifying (Dolfing et al, 1990;Altenschmidt and Fuchs, 1991;Evans et al, 1991;Schocher et al, 1991;Fries et al, 1994;Rabus and Widdel, 1995a;Hess et al, 1997), ferric iron-reducing (Lovley and Lonergan, 1990) and sulphate-reducing (Rabus et al, 1993;Beller et al, 1996) bacteria. The mechanism of anaerobic toluene activation has been elucidated recently (Biegert et al, 1996;Beller and Spormann, 1997;Leuthner et al, 1998;Rabus and Heider, 1998).…”

“…The mechanism of anaerobic toluene activation has been elucidated recently (Biegert et al, 1996;Beller and Spormann, 1997;Leuthner et al, 1998;Rabus and Heider, 1998). Other alkylbenzenes shown to be anaerobically degradable by pure cultures of denitrifying bacteria are ethylbenzene (Rabus and Widdel, 1995a;Ball et al, 1996), propylbenzene (Rabus and Widdel, 1995a) and m-xylene (Dolfing et al, 1990;Fries et al, 1994;Rabus and Widdel, 1995a;Hess et al, 1997). Anaerobic degradation of benzene (Edwards and Grbić-Galić, 1992;Lovley et al, 1995) and o-xylene (Rueter et al, 1994) under sulphate-reducing conditions and of p-xylene under nitrate-reducing conditions has been observed in enrichment cultures or sediments.…”

“…In addition to this metabolic aspect, we studied which phylogenetic types of denitrifiers were enriched by crude oil. This was done by 16S rRNA-targeted, whole-cell hybridization, a proven tool for examining, at the phylogenetic level, the composition of microbial communities in natural habitats Zarda et al, 1997), in diesel fuelcontaminated laboratory columns (Hess et al, 1997) and in a sulphate-reducing enrichment culture growing with crude oil .…”