Anaerobicaly - Composted Environmental Wastes as Organic Fertilizer and Ident...
asm slides
1. Molecular Characterization of Bacteria in Soils obtained from Petroleum
Contaminated Sites in Kaduna state, Nigeria
Presented at
115th General Meeting Of The American Society For Microbiology
May 30 – June 2, 2015
New Orleans, Louisiana
By
H. M. Raji1*, J. B. Ameh1, S. A. Ado1, S. E. Yakubu1 and A.J. Weightman2
1Dept. of Microbiology, Ahmadu Bello University, Zaria, KD, Nigeria
2School of Biosciences, Cardiff University, Wales, CF, United Kingdom
*Corresponding author: e-mail address: habibasalam19@yahoo.com
2. ABSTRACT
Culture–dependent methods of identifying bacteria present in the soil often give limited
information about their diversity owing to the fact that a large majority of these bacteria are viable
but non-culturable. Metagenomics offer a viable means of depicting microbial taxonomic and
functional diversity. Soil samples were collected from three sites exposed to petroleum products
in Kaduna state located in the north-western part of Nigeria, these sites are an auto workshop, a
trailer park, and a site close to a stream receiving effluent from a petroleum refinery. The samples
were collected from two depths (17 – 20 cm, and 37 – 40 cm respectively), and analysed based
on physico-chemical parameters, PCR amplification of the partial 16S ribosomal RNA and
phylogenetic studies. Genomic DNA was extracted from the samples in triplicates, and PCR-
DGGE conducted; the forward primer has a 40 base GC clamp attached to it to enhance analysis
with DGGE, the primer pair used is 357F-GC and 518R. Prominent bands on the DGGE gel were
excised and sequenced. The sequences obtained were analysed on Finch tv software before
subsequently subjected to BLAST analysis on the NCBI website for closest alignments.
Sequence alignment and phylogenetic analysis were carried out using the MEGA version 4.0
software. The sequences from the shallow samples from the auto workshop formed a separate
cluster from the deep samples; and are all closely affiliated to Proteobacteria. The sequences
from the trailer park soil showed closest relations to Gram positive high G+C bacteria and Gram
positive low G+C bacteria. The closest relatives to the sequences from the refinery effluent site
were members of Proteobacteria, Gram positive high G+C bacteria and Gram positive low G+C
bacteria; thus showing more diversity than the other two sites. The study showed that the depth
of sampling, anthropogenic activities as well as the soil physico-chemical parameters might play
a role on the bacterial diversity of a soil sample.
Key words: soil, bacteria, Nigeria
3. Introduction
Nigeria is a country blessed with a lot of natural resources, petroleum inclusive.
Activities relating to the drilling, refining and transportation of petroleum and its
products often leads to contamination in the environment.
The presence of these petroleum hydrocarbons in the terrestrial and/or aquatic
environment typically leads to adaptation of these hydrocarbons by indigenous
microorganisms, thus utilizing them as sources of Carbon and energy.
Bacterial bioremediation of such environments offers a cost effective and eco-
friendly solution to the hazard.
However, culture dependent methods of determining the bacterial community in the
environment are often misleading and inadequate because a large percentage of
these bacteria are not culturable thus, are not adequately represented.
Thus, Metagenomics plays a major role in determining the phylogenetic as well as
functional diversity of these hydrocarbon-degrading bacteria.
4. Aim and Objectives
The aim of this study is to determine the phylogenetic diversity of bacteria present
in soils from certain petroleum-contaminated sites in Kaduna state, Nigeria.
Objectives:
1. To determine the physicochemical characteristics of soils collected from
petroleum-contaminated sites in Kaduna state, Nigeria
2. To conduct PCR amplification and DGGE analysis of the bacterial 16S rRNA
gene in petroleum-contaminated soils using the primers, 357F-GC and 518R
3. To perform phylogenetic analysis of the prominent nucleotide sequences
obtained from the petroleum-contaminated soils.
5.
6. Materials and Methods
Soil samples: Three (3) petroleum-contaminated sites in Kaduna state (North western
region), Nigeria were the subject of this study, they are as follows: A Mechanic Workshop, a
Trailer park, and a Petroleum refinery effluent site. Two (2) samples were collected from
each site; the samples were obtained at two depths, 17 – 20 cm and 37 – 40 cm,
respectively, making a total of six samples.
DNA extraction: Genomic DNA was extracted from 0.5 gram of each soil sample in
triplicates, using the Fast DNA Spin kit for soil (MP Biomedicals, U.K), the procedure was
carried out using the manufacturer’s instructions.
PCR amplification of 16S ribosomal RNA and DGGE analysis of partial 16S rRNA gene:
Amplification of 16S rRNA was performed using the primers, 357F-GC* and 518R (Eurofins
MWG, U.K), which amplify a product of 201 bp. The GC clamp (40 bp) attached to the
forward primer is necessary to enhance separation of the bands during DGGE (30% - 60%
urea-formamide).
Sequencing of DNA from excised bands and phylogenetic analysis: Aliquots of the
reamplified amplicons were sequenced (Eurofins MWG, U.K), the resulting sequences were
cleaned by removing GC clamp sequences using Finch tv program. The BLASTN program
from the National Center for Biotechnology Information (NCBI) website was used to conduct
DNA similarity searches. Of the thirty DNA sequences sequenced, only twenty five were
successfully used for phylogenetic analysis. Multiple sequence alignment and evolutionary
analysis was carried out using the MEGA 4 program (Tamura et al., 2007).
7. Sample
Textural pH O and G
Moistur
e
Organic Nitrogen Available Ca Mg K Na
Class Content Content Carbon
Phospho
rus
USDA (mg/L) (%) mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
mg/k
g
T.D
Sandy
5.3 140 0.6 43.09 2.8 12.95 4 1.3 0.45 0.25
loam
T.S
Sandy
5.5 1 590 3.1 8.78 0.98 47.6 3.2 1.1 0.44 0.27
loam
M.D Loamy 5.8 1 220 1.4 4.79 0.06 6.48 1 0.3 0.42 0.21
M.S
Sandy
5.9 1 340 0.8 16.36 1.33 20.3 3.6 1.2 0.41 0.19
loam
R.D Loamy 5.4 770 0.75 9.58 1.05 5.43 2 0.9 0.31 0.18
R.S Loamy 5 940 1.1 10.77 1.19 6.3 2.2 0.8 0.34 0.21
Table 1: Textural Classification and Physico-chemical analysis of Petroleum-contaminated Soils
KEY:
MS: Mechanic workshop shallow; MD: Mechanic workshop deep
TS: Trailer park shallow; TD: Trailer park deep
RS: Refinery effluent shallow; RD: Refinery effluent deep
8. 0.01 0.1 1 10 100 1000 10000
Copper
Manganese
Zinc
Lead
Nickel
Cadmium
Chromium
Cobalt
Fig. 1 Heavy metal content of soils sampled from petroleum-contaminated sites in Kaduna state,
Nigeria
R.S
R.D
M.S
M.D
T.S
T.D
KEY:
MS: Mechanic workshop shallow; MD: Mechanic workshop deep
TS: Trailer park shallow; TD: Trailer park deep
RS: Refinery effluent shallow; RD: Refinery effluent deep
11. Conclusion
Heavy metals such as zinc, lead and copper were detected in high quantities in
the petroleum contaminated soils sampled in this study
The DGGE analysis of soil from Trailer park and Refinery effluent sites showed
similar profile in the two sampling depths while, the Mechanic workshop soil
had different profiles at both depths.
Phylogenetic analysis of the 16S rRNA sequences obtained from the
petroleum-contaminated soils revealed members of Alphaproteobacteria,
Gammaproteobacteria, Betaproteobacteria, Gram positive low G+C bacteria,
Gram positive high G+C bacteria and Acidobacteria
The Mechanic workshop soil indicated more bacterial diversity than the Trailer
park and Refinery effluent soils respectively.
12. References
Muyzer, G., De Waal, E.C., Uitterlinden, A.G. (1993) Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of
polymerase chain reaction-amplified genes coding for 16S rRNA. Applied
Environmental Microbiology 59: 695–700.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0.
Molecular Biology and Evolution 24:1596-1599
