Physicochemical analysis
The soil samples obtained for characterization of the spill site showed the concentration of TPH and PAHs at different depths of the soil analysed. The surface (0.0 – 0.5) sample had an extractable TPH value of 6231 mg/kg. The subsurface samples from 1m, 1.5m and 2.0m depths had extractable TPH concentration of 4836 mg/kg, 9112 mg/kg and 7273 mk/kg respectively. Results of other physicochemical analysis incuding pH, arsenic, barium, cadmium, cobalt, copper, lead, mecury, nickel, total chromium and zinc are shown in Table 1.
The concentration of polycyclic aromatic hydrocarbons was low and was only detected in the surface sample and the 1m depth samples. The surface sample had a PAH concentration of 0.13 mg/kg while the subsurface sample had PAH concentration of 1.36 mg/kg. The pristine soil TPH was 479.7mg/kg while TPH for days 0, 09, 18, 36 and 56 was 8635.68 mg/kg, 6125.7 mg/kg, 4171.9 mg/kg, 2435.2 mg/kg and 677.2 mg/kg respectively.
Microbial dynamics and abundance during remediation
A total 1,211 bacterial species was detected for all the samples. The bacterial metagenome for all the samples was dominated by Proteobacteria for both oil polluted and unpolluted soils (Fig. 1), however the unpolluted soil harboured the least Proteobacteria abundance (49.7%). The abundance of Proteobacteria continuously increased from day 0 to day 36. Proteobacteria mostly of the class Alphaproteobacteria made up 49.7% of the originally polluted soil sample prior to remediation. On day 0 Proteobacteria alone made up 65% of the soil microbiome and increased to 67%, 69% and 74% on days 09, 18 and 36 respectively. While Alphaproteobacteria dominated on days 0 and 09, a rapid increase to the dominance of Gammaproteobacteria and Betaproteobacteria was observerd on days 18 and 36. Alphaproteobacteria later dominated once more on day 56 as the oil concentration declined to below the intervention value (Fig 2.). As the dominance of Proteobacteria increased during the period of remediation, there was also a simultenous decrease in the abundance of Actinobacteria from 36% prior to intervention by Landfarming to 3.4% on day 56. Apart from Proteobacteria and Actinobacteria, other bacterial phyla’s detected includes Firmicutes, Acidobacteria, Bacteriodetes and Planctomycetes. The complete list of the detected bacterial phyla is shown in Fig. 2. The core microbiome detected during the period of remediation include Mycobacterium, Burkholderia, Rhodoplanes, Methylobacterium and Bacillus. A comparison of the crude oil polluted soils to the pristine soil showed 19 bacterial genera were differentially represented in the polluted soils and they include Clostridium, Bacillus, Staphylococcus, Lactobacillus, Parachlamydia, Phenylobacterium, Edaphobacter, Rhodoplanes, Burkholderia, Jeotgalicoccus, Desulfosporosinus, Methylobacterium, Desulfotomaculum, Acidocella, Deinococcus, Mycobacterium, Propionibacterium, Ochrobactrum, Azospirillum.
Diversity analysis following oil spill and during remediation
Analysis of the effect of the oil spill on microbial diversity revealed the presence of hydrocarbons reduced microbial diversity. Effect of hydrocarbons on bacterial diversity was inferred using Chao1 diversity index. The result of our analysis showed the polluted soil samples prior to any sort of intervention was the least diverse (Fig. 3). The hydrocarbon concentration in the soil significantly reduced bacterial diversity compared to the pristine soil which represents the soil true diversity prior to the oil spill. Following the commencement of intervention by Landfarming remediation, the diversity continued changing and increasing as the concentration of hydrocarbons in the soil reduced. Principal coordinates analysis (PCoA) saw a close clustering of the polluted soil prior to remediation and a wider spread of the polluted soil samples on the PCoA plot (Fig. 4) during Landfarming which is indicative of a continuous changing dynamic of the soil microbiome as different fractions of the hydrocarbons present in the soil were degraded.
Predictive microbial community functional response during remediation
To determine the functional responses of the bacterial community to the environmental stress caused by the presence of the crude oil spill, the GREENGENES classified bacterial species were subjected to further analysis using PICRUST. 6,909 KEGG orthologs were detected for the entire samples. The mean abundance of pathways detected was 427,917.46. The oil polluted samples were all compared to the pristine soil for differentially represented KEGG pathways. Thirty five pathways were found to be differentially represented between the pristine soil the polluted soil prior to Landfarming. The pathways were mainly pathways for hydrocarbons degradation and they include naphthalene degradation, polycyclic aromatic hydrocarbon degradation, benzoate and aminobenzoate degradation pathways among others (Fig. 6a – 6c). As remediation commenced mostly the same predicted pathways remained significantly differentially represented from day zero to day 18 after which a decline in the degradative pathways was observed. On days 36 and 56 only pathway for transporters and pathways for phosphonate and phosphinate metabolism were differentially represented.