Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure

TLDR

A most‑probable‑number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon‑degrading bacteria, addressing the inability of existing methods to distinguish these groups. The method employs separate 96‑well microtiter plates, using hexadecane for alkane degraders and a phenanthrene/anthracene/fluorene/dibenzothiophene mixture for PAH degraders, with growth detected by a color change of iodonitrotetrazolium violet after 2–3 weeks at 20 °C. The MPN procedures proved accurate and selective, yielding estimates comparable to heterotrophic plate counts for pure cultures, with no false positives from non‑degraders, and are suitable for reliable field assessment of hydrocarbon‑degrading microbial populations.

Abstract

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 degrees C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.