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

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.

Field performance of alder-Frankia symbionts for the reclamation of oil sands sites

“…PAH-degrader counts were determined in 96-well plates as described by Wrenn and Venosa (1996) except that four PAHs (10 g phenanthrene/l, 1 g anthracene/l, 1 g fluorene/l, and 1 g dibenzothiophene/l) were dissolved in hexane and the Bushnell-Haas medium was used without NaCl addition. After incubation for 3 weeks at room temperature, positive wells developed a yellow-brown colour due to PAH degradation.…”

Prepared bed bioremediation of oily sludge in an oilfield in northern China

“…Total petroleum hydrocarbon (TPH) degraders and PAH degraders were enumerated by the most-probable-number (MPN) technique [8]. The community level physiological profiling (CLPP) of the sludge microbial communities was examined using Gram-negative microplates (BIOLOG Inc.) according to the method of Garland and Mills [9].…”

Influence of flooding on growth, nitrogen availability in soil, and nitrate reduction of young oak seedlings (Quercus robur L.)

“…Denitrifying bacteria were numerated with a most-probablenumber (MPN) procedure [37] using NB Medium and Griess-Ilosvay reagent as described by Garcia et al [17]. For each sample, MPN was calculated with MPN Calculator 4.04 software using standard Mac Crady tables.…”