The use of a solid adsorber resin for enrichment of bacteria with toxic substrates and to identify metabolites: degradation of naphthalene, O-, and m-xylene by sulfate-reducing bacteria

Abstract

Anaerobic sulfate-reducing bacteria were enriched from contaminated aquifer samples with naphthalene, o-, and m-xylene as sole carbon and energy source in the presence of Amberlite-XAD7, a solid adsorber resin. XAD7 served as a substrate reservoir maintaining a constantly low substrate concentration in the culture medium. In equilibration experiments with XAD7, the aromatic hydrocarbons needed up to 5 days to achieve equilibrium between the water and the XAD7 phase. The equilibrium concentration was directly correlated with the amount of added substrate and XAD7. In the enrichments presented here, XAD7 and aromatic hydrocarbons were adjusted to maintain substrate concentrations of 100 microM m-, or o-xylene, or 50 microM naphthalene. After five subsequent transfers, the three cultures were able to grow with higher substrate concentrations in the absence of XAD7 although they grew best with lower hydrocarbon concentrations. Two new xylene-degrading cultures were obtained that could not utilise toluene as carbon source. O-xylene was degraded anaerobically by a culture, which could also oxidise m-xylene but not p-xylene. Eighty-three percent of the electrons from o-xylene oxidation were recovered in the produced sulfide, indicating a complete oxidation to CO2. Another sulfate-reducing enrichment culture oxidised m-xylene completely to CO2 but not o-, or p-xylene. A naphthalene-degrading sulfate-reducing enrichment culture oxidised naphthalene completely to CO2. Metabolites of naphthalene degradation were recovered from the XAD7 phase and subjected to GC/MS analysis. Besides the metabolites 2-naphthoic acid and decahydro-2-naphthoic acid which were identified by the mass spectrum and coelution with chemically synthesised reference compounds, the reduced 2-naphthoic acid derivatives 5,6,7,8-tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. Cultivation of bacterial cultures in the presence of XAD7 and subsequent derivatisation and extraction of metabolites directly from the solid XAD7 resin provides a new method for the isolation of sensitive bacteria and identification of metabolites.