Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid Southwestern soils

Abstract

Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R(2)A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.

FIG. 1.

Phylogenetic tree was based on the 16S rRNA gene sequence from microorganisms representing biosurfactant producers isolated in this study as well as known biosurfactant producers from the literature. The unrooted tree was created by using the neighbor-joining method. CFB, Cytophaga-Flexibacter-Bacteroides.

FIG. 2.

REP analysis of all the isolates that matched with B. subtilis. Lane 1, strain MA12; lane 2, strain HAZ2; lane 3, strain HAZ14; lane 4, strain WP1-21; lane 5, GA1-5; lane 6, strain BHP6-1; lane 7, strain BZ15; lane 8, strain STB29; lane 9, molecular weight marker III; lane 10, 100-bp marker.