Molecular Diversity of Microbes with Probable Degradative Genes in Agricultural Soil Contaminated with Bonny Light Crude Oil

Incidence of environmental pollution due to high rate of petroleum related activities in Nigeria and other oil producing areas of the world has been associated with frequent oil spills, especially through oil wells blow out, tanker accidents and bunkering. Disasters arising from such incidence results in the discharge of crude oil into the environment affecting both soil, air and water bodies. This threatens human health and that of organisms that are dependent on soil. Soil contains a variety of microorganisms including bacteria that can be found in any natural ecosystem. Microbial survival in polluted soil depends on intrinsic biochemical and structural properties, physiological and genetic adaptation including morphological changes of cells as well as environmental modifications [1]. Over the years, isolation and identification of hydrocarbon-degrading microorganisms have been carried out using isolation techniques. Previous studies on population dynamics showed that bacteria genera such as Pseudomonas, Bacillus, Brevibacterium, Corynebacterium, Acinetobacter and Mycobacterium are potential organisms for hydrocarbon degradation [2-4]. Shi et al. [5] compared culture-based diversity of agricultural soil communities with diversity obtained by molecular means and found that molecular methods revealed a much higher bacterial diversity than classical isolation techniques.


Introduction
Incidence of environmental pollution due to high rate of petroleum related activities in Nigeria and other oil producing areas of the world has been associated with frequent oil spills, especially through oil wells blow out, tanker accidents and bunkering. Disasters arising from such incidence results in the discharge of crude oil into the environment affecting both soil, air and water bodies. This threatens human health and that of organisms that are dependent on soil. Soil contains a variety of microorganisms including bacteria that can be found in any natural ecosystem. Microbial survival in polluted soil depends on intrinsic biochemical and structural properties, physiological and genetic adaptation including morphological changes of cells as well as environmental modifications [1]. Over the years, isolation and identification of hydrocarbon-degrading microorganisms have been carried out using isolation techniques. Previous studies on population dynamics showed that bacteria genera such as Pseudomonas, Bacillus, Brevibacterium, Corynebacterium, Acinetobacter and Mycobacterium are potential organisms for hydrocarbon degradation [2][3][4]. Shi et al. [5] compared culture-based diversity of agricultural soil communities with diversity obtained by molecular means and found that molecular methods revealed a much higher bacterial diversity than classical isolation techniques.
A variety of molecular methods have been developed to assay the presence of micro-organisms in soil. Most recently, the method of choice to determine what micro-organisms are present in environmental sample is to amplify the conserved small subunit rRNA gene; where DNA is isolated from the soil using bead beating and Polymerase Chain Reaction (PCR) with universal or gene-specific primers used to amplify the specific gene from the sample. This study looked at the diversity of microorganisms persistent in agricultural soil sample polluted with 100 ml of 100% Nigerian Bonny light crude oil and left for four years with a view to ascertain the presence of microbes with probable degradative gene for crude oil degradation which can be harnessed for the creation of superbugs for faster clean up opertaions and to confirm similarities in microbial identities.

Procurement of samples
The crude oil used was bonny light Crude and was collected with sterile containers from Akiri in Oguta, Imo State, Nigeria. The agricultural soil subjected to pollution was obtained from Federal University of Technology Owerri (FUTO) farm land using surface sterilized soil auger at the depth of 15-30 cm.
of Yee et al. [6]. The setup was kept in a chamber for a period of four years with a light cycle of 11 h darkness and 13 h light.

Molecular analysis
Molecular analysis was performed at the GS FLX Titanium Sequencing Service company-Inqaba, South Africa. Methodology was based on PCR and metagenomics analysis.

DNA extraction from soil sample
DNA extraction form soil sample was performed using ZYMO soil DNA extraction kit according to the manufactures. According to the method, genomic DNA was extracted by weighing out 0.25 grams of soil sample using an analytical Balance. The sample was the added into a ZR Bashing Bead TM Lysis Tube followed by the addition of 750 µ Lysis Solution to the tube. The content of the 2 ml tube disrupted by mixing in a vortex mixer at maximum speed for 5 minutes. The ZR Bashing Bead TM Lysis Tube was centrifuged in a micro centrifuge at ≤10,000 xg for 1 minutes. 400 µl of the filtrate was added to a Zymo-Spin TM IV Spin Filter in a Collection Tube and centrifuge at 7,000 rmp (˜7,000 xg) for 1 minutes. This was followed by the addition of 1,200 µl of soil DNA Binding Buffer to the filtrate in the Collection Tube. 800 µl of the mixture from above was added to a Zymo-Spin TM IIC Column in a Collection Tube and centrifuge at 10,000 xg for 1 minute. Flow through from the collection tube was discarded and this particular step was repeated with the remaining filtrate. 200 µl of DNA Pre-Wash Buffer was thereafter added to the Zymo-Spin TM IIC Column in a new collection tube and centrifuged at 10,000 xg for 1 minute. Thereafter, 500 µl of Soil DNA Wash Buffer was added to the Zymo-Spin TM IIC Column and centrifuged at 10,000 xg for 1 minute. The Zymo-Spin TM IIC Column was transferred into a clean 1.5 ml micro centrifuge tube and 100 µl of DNA Elution Buffer added directly to the column matrix. This was centrifuged at 10,000 xg for 30 seconds to elute the DNA. The eluted DNA was transferred into a filter unit of Zymo-Spin TM IV-HRC Spin Filter in a clean 1.5 ml micro centrifuge tube and centrifuged at exactly 8,000 xg for 1 minute. The filtered DNA was then used for PCR and DNA sequencing.

Polymerase Chain Reaction [PCR]
The PCR was carried out in a 20 µl reaction mixture containing a 5X HOT FIRE Pol blend master mix (ready to use) composed of FIREPol ® DNA polymerase Proof-reading enzyme, 5X reaction buffer, 7.5 mM MgCl 2 , 1 mM dNTPs of each have 200 µM of dATP, dCTP, dGTP, dTTP. A combination of 4 µl of master mix, 0.2 µl each of forward and reverse 16S rRNA primer and 2 µl of template gDNA constituted 6.4 µl. Hence 13.6 µl of sterile distilled water was added to make it up to the recommended PCR reaction mix of 20 µl .The entire mixture was then vortexed and loaded together with positive and negative control (dH 2 0) into the thermal cycler (eppendorf vapor protect). The PCR reaction was carried out with an initial denaturation at 95 o C for 5 min, followed by 30 consecutive cycles at 95 o C for 30 sec, and annealing temperature of 55 o C for 1 minute and then 72 o C holding for 1 minute. This was then followed by a final extension step at 72 o C for 10 minute.

DNA Sequencing
DNA sequencing was performed by Next Generation Sequencing Technique to determine the nucleotide sequence of all microorganisms present in the soil sample using automated PCR cycle-Genome Sequencer TM FLX System from 454 Life Sciences TM and Roche Applied. Sequence analysis and alignment was performed using Vector NTI suite 9 (InforMax, Inc.) and the resulting nucleotide sequences were compared to sequences obtained from GenBank 1 by BLASTx. Analysis using CLO Bio software as well as BLASTn 2 using NCBI. For every sample set, every read was BLASTED and the result file saved. The top 5 hits for every BLAST result (i.e., species name) was counted and a record was kept of how many times each species appeared as a hit. The number in the last column basically is the number of times a read hit/ matched to that species. The frequency (i.e., count/total number of reads) and absolute count of each species were reported and used to name the specific organism (Table 1).

Results and Discussion
The study of identification of bacteria for the biodegrading capabilities is important in microbial ecology, especially with molecular techniques. In particular, analysis of the microbial communities that take part during in-situ hydrocarbon biodegradation activities has been a challenge to microbiologist. Interest in this area has been catalyzed by the rapid advancement of molecular ecological methodologies. Thus, the ability of an organism to degrade a specific substrate and persist within such environment is clear evidence that its genome harbored the relevant degrading gene [7]. The previous studies by Bindu and Satish [8] and Jyothi et al. [9] on hydrocarbon degradation by bacteria reveal that catechol 2, 3 dioxygenase is one of the enzyme involved in hydrocarbon degradation.
Molecular confirmation of similarities in microbial Identities was obtained by creating different dendrograms/distance trees. Gene sequencing carried out read 513 different nucleotide sequences. Every read was BLASTED and the result file saved. The report on the frequency of reads of each species is as shown in Table 1. Seven phylum with 47 corresponding culture-dependent species and 169 culture-independent bacteria clone was obtained. The resultant haplotree/cladogram however, showed clades of proteobacteria (b-and g-proteobacteria), bacteria/enterobacteria, firmicutes, plantomycetes, acidobacteria group/ fibrobacteres, Bacteriodetes/chlorobi, Actinobacteria/high G+C and chloriflexi phyla ( Figure 1). The nucleotide sequences with no hit was sent to Genbank for asigning of ascension number. The isolation of the aforementioned organisms from crude oil polluted agricultural soil left for four years, depict that the organism probably, have degradative genes coding for enzymes for hydrocarbon catabolism which aided their survival. These have been confirmed by the presence of plasmid DNA in culture -dependent isolates obtained and published elsewhere [10].

Taxonomical classification and percentage diversity
Further taxonomical analysis was carried out with reads of sufficient Q scores (>q30) and lengths and a total of 420 read count of top kingdom classification of 100% bacteria kingdom was obtained. Top phyla classification depict that that Proteobacteria had the highest diversity of 57.14% followed by Acidobacteria (20.24%), Unknown (16.67%), Firmicutes (3.33%), Bacteroidetes (2.38%), and Planctomycetes (0.24%) in that order ( Figure 2). Top class and order classification of phylum proteobacteria, class beta proteobacteria and order Burkholderiales also had similar highest values of 53.81% ( Figure  3 and Figure 4) whereas in top family classification, Burkholderiaceae recorded the lowest diversity of 0.24% ( Figure 4). Furthermore, the family of unknown increased by 2.38% while diversity of Acidobacteria phyla, class, order and family remained constant (20.24%). Generally, the dendogram of the BLAST hit showing resultant clades with their leaves and height of the branch points indicating the similarity and differences of isolates from each other (the greater the height, the      greater the difference) as well as the percentage diversity of all the taxonomical classification of bacterial isolates from the BLAST output result based on hits and non-hit read counts are shown in Figure 6 and Figure 7 respectively. The result ( Figure 5) depict that about 33.12% had no hit.
Indeed, microbial degradation is the major and ultimate natural mechanism by which one can clean up the petroleum hydrocarbon pollutants from the environment [10,11]. The recognition of biodegraded petroleum hydrocarbons in the environment as observed in previous studies [2,3,9,12,13], which was evident through detectable biodegradation of n-alkane profile of the crude oil by microorganisms supports the findings of this study. The microbial genera, namely, Arthrobacter, Alcaligenes, Burkholderia, Mycobacterium, Micrococcus, Pseudomonas, Acinetobacter, Bacillus, Sphingomonas, Corynebacterium and Rhodococcus have been incriminated to be involved in hydrocarbon degradation as observed in the percentage diversity of the taxonomical classification in this study; as these organisms fall within similar identified phyla, class, order and family of bacterial isolate during this metagenomic analysis. From the findings of previous studies and in   line with this study, bacteria are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in the environment having in them enzymes for hydrocarbon degradation. This corroborates the report made by Rahman et al. [14] and Brooijmans et al. [15] who studied on hydrocarbon degrading bacterial in petroleum sludge. The persistence of the identified bacterial isolates in this study could also be due to the ability of the isolates to produce bio surfactants which aids in the formation of micelles to enhance uptake of hydrocarbons. Studies have also shown that total bacteria population in polluted soil are more than that in unpolluted soil [16][17][18] which implies that those organisms are the active degraders of that oil.
Metagenomic analysis carried out in this study have actually helped in detection of Acidobacteria phyla which are under-represented in culture even though they are physiologically diverse and ubiquitous as well as so many uncultured genera. The low diversity of Planctomycetes is not surprising since they are aquatic bacteria phyla and are found in samples of brackish, marine and fresh water. Further molecular studies are therefore needed to detect specific catabolic genes resident in these hydrocarbon degrading isolates. This can help to produce superbugs required for faster remediation, cost effective and efficient bioremediation protocol for Nigerian oil polluted soil.

Conclusion
The isolation of the aforementioned organisms from crude oil polluted agricultural soil left for four years, depict that the organism probably, have degradative genes which aided their survival.