DIVERSITY OF BACTERIA IN A PETROLEUM-CONTAMINATED SOIL SPIKED WITH POLYCYCLIC AROMATIC HYDROCARBONS

Presented atThe 39th Annual Scientific Conference and General Meeting of The

Nigerian Society for MicrobiologyBy

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

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

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

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

Aim:This study aimed at determining the possible effect the presence of PAHs has on the diversity of bacteria in a petroleum-contaminated soil

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

Shannon diversity index (H):H = -sum (Piln[Pi]) (natural log)S = sum of the different species presentE = H/ln(S) (natural log)(Pi: proportion of each specie based on the total number of species; In[Pi]: natural log of Pi)

RESULTS

Plate I: Amplification of 16S rRNA gene in a Mechanic workshop soil treated with polycyclic aromatic hydrocarbons

M: Molecular markerMS: Untreated Mechanic workshop shallow MD: Untreated Mechanic workshop deepMSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop deepMSp: Phenanthrene-spiked Mechanic workshop shallow MDp:Phenanthrene-spiked Mechanic workshop deepMSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deepMSb: B[a]P-spiked Mechanic workshop shallow MDb: B[a]P-spiked Mechanic workshop deep+ve: Positive control -ve: Negative control

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 = 2.8292KEY:MS: Untreated Mechanic workshop shallow MD: Untreated Mechanic workshop deepMSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop deepMSp: Phenanthrene-spiked Mechanic workshop shallow MDp:Phenanthrene-spiked Mechanic workshop deepMSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deepMSb: B[a]P-spiked Mechanic workshop shallow MDb: B[a]P-spiked Mechanic workshop deep

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

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)

1924

99

3497

3995

66

1929

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 deepMSa: Acetone-treated Mechanic workshop shallow MDa: Acetone-treated Mechanic workshop deeMSp: Phenanthrene-spiked Mechanic workshop shallow MDp: Phenanthrene-spiked Mechanic workshop deepMSc: Chrysene-spiked Mechanic workshop shallow MDc: Chrysene-spiked Mechanic workshop deepYellow 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

untreated soil

control Phe. Chry. Bap0%

10%

20%

30%

40%

50%

Treatments in soil sample

Percentage abundance

Figure 2: Distribution of bacterial taxonomic groups in a soil spiked with polycyclic aromatic hydrocarbons

Proteo

bacte

ria

Firmicu

tes

Actinob

acteri

a

Chlorofl

exi

Uncultu

red ba

cteria

0%10%20%30%40%50%

Bacterial phyla

Percentage abundance

Figure 3: Abundance of bacterial species in a soil spiked with polycyclic aromatic hydrocarbons

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%)

ACKNOWLEDGEMENTS:

Petroleum Technology Development Fund (P.T.D.F), Nigeria Science and Technology Education Post Basic (STEP-B) Fund,

Ministry of Education, Nigeria

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