Influence of Biostimulation Treatment Using Composted Plant Biomass on Bacterial Diversity of an Aged Petroleum Contaminated Soil as Determined by Culture-dependent and 16S rRNA Gene PCR-DGGE Based Identification Methods

Abstract

Influence of biostimulation treatment using composted plant biomass on bacterial diversity of an aged crude oil contaminated soil (ACOCS) was determined using culture-dependent and 16S rRNA gene PCR-DGGE based identification methods. Seven treatment plots were designed and included treatments A (TPA) through G (TPG). Samples were collected bi-weekly from 7 treatment plots designed in situ during a 70-day study period that spanned 10 weeks. Composted (2,500 g each) Water hyacinth (EC), Mexican sunflower (TD) and Bermuda grass (CD) were used as nutrient supplements in 4,000 g of ACOCS in situ. TPA was un-amended while TPB, TPC, and TPD had EC, TD and CD added singly. TPE had EC and TD while TPF contained EC and CD in combination. TPG consisted of EC, TD and CD combined. Bacterial isolates were obtained on mineral salts medium and identified based on their morphological and biochemical characteristics. DGGE fingerprints of PCR-amplified 16S rRNA bacterial gene fragments were also determined using the universal primer set: 7F: 5’-GAGTTTGATCCTGGCTCAG-3’ and 1492R:5’-GGTTACCTTGTTACGACT-3’that corresponded to positions 968 and 1401 of Escherichia coli 16S rRNA gene sequence. DGGE bands fell into corresponding operational taxonomic units based on a threshold of 91-100% sequence similarity. Dendrogram showed dominant DGGE bands in TPB though TPG over time with TPA having no band. The isolates are known crude oil utilizers and are closely related to Gordonia sp. BS261404 with 98% sequence similarity, Aquitalea magnusonii KG26145 (96%), Sphingobacterium sp. K1261411 (97%) and Achromobacter sp. HQ261417 (100%). Data indicated that pseudomonads are the dominant bacteria involved in hydrocarbon biodegradation after biostimulation with the composted plant materials. Cultural and molecular methods of cultivation of microorganisms are neither contradictory nor excluding and should be considered as complementary to interrogate the bacterial diversity in the natural soil environment.