Numerical simulation of bioremediation of poly aromatic hydrocarbon polluted
nsm presentation 2016
1. DIVERSITY OF BACTERIA IN A PETROLEUM-CONTAMINATED SOIL
SPIKED WITH POLYCYCLIC AROMATIC HYDROCARBONS
Presented at
The 39th Annual Scientific Conference and General Meeting of The
Nigerian Society for Microbiology
By
Raji1*, H.M., Ameh1, J.B., Ado1, S.A., Yakubu1, S.E., Webster2, G. and
Weightman2, A.J.
1Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria
2 Cardiff School of Biosciences, Cardiff University, Cardiff, Wales,
United Kingdom
*Corresponding author: habibasalam19@yahoo.com, 0803 596 0039
2. Polycyclic aromatic hydrocarbons (PAHs ) are compounds
composed of three or more fused benzene rings.
Generally, the more the number of benzene rings, the more
recalcitrant PAHs are. PAHs are potentially carcinogenic and
mutagenic.
Microbial degradation of PAHs offers a cost-effective and eco-
friendly means of removing them from the environment (Afzal et
al., 2011; Korotkevych et al., 2011; Eziuzor and Okpokwasili,
2013)
INTRODUCTION
3. A large percentage of soil bacteria capable of degrading these
compounds are underrepresented on laboratory media as such
their metabolic and functional capabilities are not adequately
studied using culture-dependent methods
Metagenomics provides a wholesome approach in analysing
microbial communities (Chikere, 2013; Gohedja et al., 2014)
The conserved nature of the 16S rRNA gene makes it an
excellent index for taxonomic studies
4. Denaturing Gradient Gel Electrophoresis (DGGE) is a commonly
applied method of studying bacterial communities using PCR
amplicons of the 16S gene
DGGE is especially useful in fingerprinting complex microbial
communities during degradation of pollutants in the environment
(Hong et al., 2007; Korotkevych et al., 2011; Festa et al., 2013)
The presence of PAHs in the soil could affect the metabolic and
enzymatic capabilities of the bacterial community thus affecting
their diversity
5. Aim:
This study aimed at determining the possible
effect the presence of PAHs has on the diversity
of bacteria in a petroleum-contaminated soil
6. MATERIALS AND METHOD
Collection of soil samples
Spiking of soil samples with PAHs (phenanthrene, chrysene
and benzo[a]pyrene)
Extraction of DNA
Polymerase Chain Reaction and Denaturing Gradient Gel
Electrophoresis (PCR-DGGE) analysis of 16S rRNA
amplicons
Alignment and phylogeny of 16S rRNA sequences using
MEGA 4 software
7. Shannon diversity index (H):
H = -sum (Piln[Pi]) (natural log)
S = sum of the different species
present
E = H/ln(S) (natural log)
(Pi: proportion of each specie based
on the total number of species; In[Pi]:
natural log of Pi)
9. Plate I: Amplification of 16S rRNA gene in a Mechanic workshop soil treated with polycyclic aromatic
hydrocarbons
M: Molecular marker
MS: Untreated Mechanic workshop shallow MD: Untreated Mechanic workshop deep
MSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop deep
MSp: Phenanthrene-spiked Mechanic workshop shallow MDp:Phenanthrene-spiked Mechanic workshop deep
MSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deep
MSb: B[a]P-spiked Mechanic workshop shallow MDb: B[a]P-spiked Mechanic workshop deep
+ve: Positive control -ve: Negative control
10. Plate II: DGGE profile of amplified 16S rRNA from Mechanic workshop soil treated with PAHs (bands excised for
sequencing are as indicated); Shannon Index of diversity = 2.8292
KEY:
MS: Untreated Mechanic workshop shallow MD: Untreated Mechanic workshop deep
MSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop deep
MSp: Phenanthrene-spiked Mechanic workshop shallow MDp:Phenanthrene-spiked Mechanic workshop deep
MSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deep
MSb: B[a]P-spiked Mechanic workshop shallow MDb: B[a]P-spiked Mechanic workshop deep
11. Mechanic workshop
Shannon Weiner index (H) 2.8292
Species richness (S) 140
Evenness (E) 0.5725
Table 1: Shannon Index of Diversity of Bacteria in a Petroleum-contaminated Soil
12. MS Acinetobacter variabilis strain NIPH
MSa Acinetobacter variabilis strain NIPH
MSa Acinetobacter sp. IHBB 9150
MS Bacterium JNKLA9
MS Pantoea sp. Iso10-19
MS Salmonella enterica
MS Cronobacter sakazakii strain NCTC 815
MDa Uncultured bacterium gene for clone:SK27B-25
MDp Uncultured bacterium gene for clone:SK27B-25
MDc Uncultured bacterium clone BiphS1 35
MDa Uncultured Chloroflexi bacterium clone
MD Uncultured bacterium clone SF-183
MSc Bacillus subterraneus strain MD-A13
MSa Uncultured bacterium clone S11-129
MSc Bacillus sp. THG-HS227
MD Bacillus sp. THG-HS227
MSc Uncultured bacterium gene for clone: TSNIR003_L13
Pseudomonas putida(D37923.1)
Bacillus firmus(AJ491843.1)
Geitlerinema sp. (U96442.1)
19
24
99
34
97
39
95
66
19
29
41
86
83
44
64
99
64
Fig. 1: A Maximum Parsimony Tree showing Phylogenetic Affiliation of 16S rRNA sequences obtained from a Mechanic
workshop soil in Kaduna state.
The tree was rooted with a genus from the Cyanobacteria group (Geitlerinema sp.); reference strains included in the tree
are indicated in bold type. Bootstrap values carried out for 500 replicates; the numbers at the nodes indicate the bootstrap
value in percent.
MS: Untreated Mechanic workshop shallow MD: Untreated Mechanic workshop deep
MSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop dee
MSp: Phenanthrene-spiked Mechanic workshop shallow MDp: Phenanthrene-spiked Mechanic workshop deep
MSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deep
Yellow highlight: Shallow sampling sites (17 – 20 cm) Red highlight: Deep sampling sites (37 – 40 cm)
P: Proteobacteria; C: Chloroflexi U: Uncultured bacteria; F: Firmicutes
P
U
C
F
15. CONCLUSION
The closest matches to the 16S sequences based on
similarity search on the NCBI website belong to the
following bacterial phyla: Protebacteria, Firmicutes
and Chloroflexi.
The unspiked (46%) and control soil samples (27%)
had relatively more bacteria compared to the soils
spiked with PAHs (0% -19%).
Soil samples spiked with chrysene had the highest
percentage of identified bacteria while the samples
spiked with benzo[a]pyrene had none (0%)
16. ACKNOWLEDGEMENTS:
Petroleum Technology Development Fund (P.T.D.F), Nigeria
Science and Technology Education Post Basic (STEP-B) Fund,
Ministry of Education, Nigeria
