Occurrence of hydrocarbon-degrading nitrogen-fixing bacteria in the rhizosphere of Paspalum vaginatum Sw.

Abstract

Several nitrogen-fixing and hydrocarbon utilizing bacteria were isolated from the rhizosphere of the turf grass Paspalum vaginatum Sw. using four different hydrocarbons; crude oil, decane, diesel and engine oil as substrates. The isolates showed between 98%-99% 16S rRNA gene similarity with species of Alcaligenes, Brevundimonas and Klebsiella. Only Alcaligenes faecalis C1B, Klebsiella varicola C3B and Klebsiella varicola C3BB had the nifH gene; and the genes responsible for nitrogen-fixing abilities of all the isolates were not plasmid borne. Three of the isolates, Brevundimonas diminuta C4B, Alcaligenes faecalis B5 and Alcaligenes faecalis D4A with the ability to grow well in crude oil as carbon and energy source were selected for further studies. Alcaligenes faecalis B5 had the highest growth rate constant of 0.038 µ-1 with a mean generation time of 18.46 h. Analysis of culture extracts by gas chromatography with flame ionization detector showed that Alcaligenes faecalis B5 degraded 90.5% of crude oil in 24 days, while Alcaligenes faecalis D4A and Brevundimonas diminuta C4B biodegraded 84.6% and 82.1% of the oil respectively. Confirmation of nitrogen fixing ability was determined by the acetylene reduction assay (ARA). Alcaligenes faecalis B5 produced the highest amount of ethylene (15.18 nmol) after 24 h of incubation, while Alcaligenes faecalis D4A and Brevundimonas diminuta C4B produced 12.10 nmol and 9.60 nmol of ethylene respectively. Nitrogen-fixing hydrocarbon utilizers are therefore potential seeds in the bioaugmentation of nitrogen-limited hydrocarbon-polluted environments.