CHARACTERIZATION AND MOLECULAR DIVERSITY OF SOME SOIL PROKARYOTES WITH POTENTIALS FOR POLYCYCLIC AROMATIC HYDROCARBON DEGRADATION
CHARACTERIZATION AND MOLECULAR DIVERSITY OF SOME SOIL PROKARYOTES WITH POTENTIALS FOR POLYCYCLIC AROMATIC HYDROCARBON DEGRADATION
No Thumbnail Available
Date
2016-02
Authors
RAJI, HABIBA MUSTAPHA
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
This research was focused on using molecular tools to identify and determine the diversity of soil bacteria in petroleum-contaminated sites in some parts of Kaduna State, Nigeria. Soil samples were obtained from three sites with a history of petroleum hydrocarbon contamination in Kaduna state – a mechanic workshop in Zaria that has been in operation for at least thirty years, a trailer park in Zaria where trailers are serviced and repaired, and a site close to a stream receiving effluent from the petroleum refinery in Kaduna. The soils were collected from two different depths (17 – 20 cm, and 37 – 40 cm); and subjected to physico-chemical analyses and DNA extraction. Amplification of the 16S rRNA gene was carried out using the universal primers, 357F and 518R. Amplicons of the 16S ribosomal RNA gene from the soil samples were further analysed to obtain the different nucleotide sequences using denaturing gradient gel electrophoresis (DGGE), a forty base pair (40 bp) GC clamp was attached to the forward primer to aid separation during DGGE analysis. The prominent bands on the DGGE gel were excised and sequenced. Based on the similarity search conducted on the NCBI website, the identities of the bacteria in the soils were closely affiliated to the following phylogenetic groups, Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria, Acidobacteria. Quantification of the bacterial and archaeal 16S gene in the soils by Real-time PCR using SYBR-Green fluorescence revealed that the bacterial 16S gene was more abundant than the archaeal 16S gene. The second phase of the experiment involved spiking the soil samples with PAHs having three, four and five benzene rings respectively. Genomic DNA was extracted from the spiked soil samples and used for phylogenetic studies and Real-time PCR ampification. The effect of the PAHs on the indigenous bacteria in the soils was
xxiv
determined using the Shannon diversity index, phylogenetic analysis and quantification of the bacterial 16S rRNA gene. Soils from the Mechanic workshop and Trailer park sites had similar diversity indices however; the former had a slightly higher value. Thus, making it the site with the highest diversity of bacterial species. The closest relatives of the 16S sequences from the Refinery effluent site were all species belonging to the genus, Bacillus. On the other hand, the sequences from the Mechanic workshop and Trailer park were closely affiliated to a wide variety of bacterial groups such as, Gammaproteobacteria, Firmicutes, and to a less extent, Actinobacteria and Chloroflexi. The Trailer park soil had two sequences that had high similarity to the sequences of two novel strains in the Genbank. A good number of the bacteria identified in the Refinery effluent and Trailer park sites were obtained from the soils spiked with polycyclic aromatic hydrocarbons. This was not the case with the Mechanic workshop site, as most of the bacteria identified were from the untreated soil samples. Quantification of the 16S ribosomal RNA gene in the soils spiked with PAHs revealed higher gene copies in the treated soils than the soils that were not spiked with polycylic aromatic hydrocarbons. The Mechanic workshop site had the highest number of 16S gene copies and it was observed in the soil spiked with chrysene. Thus, chrysene likely favoured the proliferation of bacteria in the Mechanic workshop soil. Culture methods should not be solely relied on for the identification of PAH-utilizing bacteria in soils since most of these species do not grow on laboratory media. Metagenomics is a powerful tool in studying the ecology of the bacterial community, as well as in the determination of the microbial functional and phylogenetic diversity. Bioremediation studies in petroleum-polluted regions in Nigeria should focus on both culture and culture-independent methods in order to fully harness the potentials of hydrocarbon-degrading bacteria.
Description
A THESIS SUBMITTED TO THE SCHOOL OF
POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY IN PARTIAL
FULFILMENT FOR THE AWARD OF PH.D MICROBIOLOGY
DEPARTMENT OF MICROBIOLOGY
FACULTY OF SCIENCE
AHMADU BELLO UNIVERSITY, ZARIA
Keywords
CHARACTERIZATION,, MOLECULAR DIVERSITY,, SOIL PROKARYOTES,, POTENTIALS,, POLYCYCLIC AROMATIC,, HYDROCARBON DEGRADATION,