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

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

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.

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.

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).

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

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

KEY:

M: Molecular marker

Lanes 1 – 3: MD (Mechanic workshop

deep)

Lanes 4 – 6: MS (Mechanic workshop

shallow)

Lanes 7 – 9: TD (Trailer Park deep)

Lanes 10 – 12: TS (Trailer park shallow)

Lanes 13 – 15: RD (Refinery effluent

deep)

Lanes 16 – 18: RS (Refinery effluent

shallow)

MS3: Acinetobacter baumannii strain-X2A [KJ806334.1]

MS4: Acinetobacter sp. MU02 [KF261600.1]

MS5 : Acinetobacter baumannii strain C-X2A [KJ806334.1]

MS1: Acinetobacter radioresistens [LM994723.1]

MS6: Acinetobacter sp. MU02 [KF261600.1]

MS2: Pseudomonas aeruginosa strain B [HE862283.1]

MD8: Burkholderia sp. RPE64 [AP013059.1]

MD11: Burkholderia sp. RPE64 [AP013059.1]

MD12: Burkholderia cenocepacia strain DWS 37E-2 [CP007781.1]

MD10: Burkholderia cenocepacia strain DWS 37E-2 [CP007781.1]

MD7: Burkholderia sp. 6hN46 [KJ879969.1]

MD9: Uncultured Burkholderia sp. Clone AG10B [JF522220.1]

RS22: Alpha proteobacterium Photinia_bac [JQ715617.1]

RS24: Rhodopseudomonas sp. JA640 [FN995103.1]

RD27: Uncultured Acidobacteria bacterium clone GYs-1-81 [JX493104.1]

RD29: Uncultured soil bacterium clone 80-d [KM205480.1]

TS13: Uncultured bacteria, clone McL 103 [FN567942]

TS14: Uncultured Bacillus sp., clone SBK22 [KM108632.1]

TD20: Bacillus sp. THG-S3 [KJ810591.1]

RD28: Uncultured Bacillus sp. Clone BB98 [KJ473580.1]

TD18: Uncultured Actinobacterium clone [JF989586.1]

TD21: Uncultured Actinobacterium clone [KJ662631.1]

RS26: Micrococcus endophyticus strain KMGL 1309-LM7 [KF740558.1]

TS16: Arthrobacter ramosus strain F34 [KM019839.1]

RS25: Uncultured bacterium, clone: CGL-UB-EUB 28D3-9 [AB583830.1]

98

45

100

52

98

88

100

91

75

95

49

53

82

90

94

96

95

0.02

Fig. 2: Neighbour-Joining Tree showing Phylogenetic Affiliation of 16S rRNA sequences obtained from 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

Yellow highlight: Shallow sampling sites (17 – 20 cm) Red highlight: Deep sampling sites (37 – 40 cm)

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.

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

Acknowledgements: