Artisanal refining is the leading source of hydrocarbon pollution in the Niger Delta with severe consequences on farmlands, fishing settlements, marine biodiversity and key biogeochemical processes. Due to slow natural recovery processes (possibly the result of unfavourable environmental conditions), most artisanal refining sites remain highly polluted even after several years of abandonment. This study, therefore, investigated the natural response of microbial communities to the presence of hydrocarbons in an artisanal refining site and how nutrient addition and intermittent tillage affects microbial alpha diversity, community structure, potential function and the overall rate of hydrocarbon sequestration.
The availability of nutrients has been demonstrated to influence the rate of hydrocarbon biodegradation [9, 40]. In this study, nutrient and oxygen availability appeared to be one of the rates limiting factors that affect biodegradation and thus likely explains the slow rate of bioattenuation in the oil-polluted site. The application of poultry droppings resulted in a significant increase in soil pH, total nitrogen, total phosphorus and total potassium. This finding implies that these soil chemical parameters were the most responsive to the remedial approach. Soil pH, nitrogen, phosphorus and potassium are important chemical factors that exert a strong influence on both the relative abundance and structure of bacterial communities . In hydrocarbon impacted soils, slightly acidic to alkaline pH (6.5–8.0) is considered optimal for biodegradation , therefore, the improved rate of hydrocarbon degradation corresponds to the mean increase in the soil pH during remediation (Table 2). Furthermore, there is evidence that the influence of pH on bacterial community structure and abundance has a corresponding effect on the availability of soil nutrients including nitrogen, phosphorus and potassium . Notably, pH significantly correlated positively with nitrogen and phosphorus, suggesting that these soil chemical features were directly impacted by a significant change in the soil pH during remediation.
In this study, 16S rRNA metabarcoding was used to investigate the diversity of microbial communities across vertical samples of the artisanal refining site and during remediation of the crude oil impacted site. A higher number of unique OTUs were present in the site pre-mediation than during remediation. Similarly, the number of unique OTUs were higher at the surface soil of the site compared to the subsurface, though the high number of shared OTUs across vertical samples suggest that conditions were not largely different across depths. These observations indicate that the intermittent tillage and addition of nutrients to the polluted site significantly impacted bacterial species diversity (diversity decreased) while the high concentration of hydrocarbons across vertical samples created a seemingly common condition irrespective of soil depth. Furthermore, the observed significant decrease in the proportionality of bacterial species during remediation suggest that improved aeration through tillage and the supply of nutrients may have reduced the dominance of some category of bacterial species as diversity indicators inversely correlated with nitrogen, phosphorus and potassium. Similar to our findings, Bonaglia et al.  reported that bacterial alpha diversity of a PAH contaminated sediment was higher at timepoint zero than during remediation irrespective of treatment-type.
Comparison of the bacterial community structure pre-remediation and during remediation revealed significant (p < 0.05) differences. Significant changes in the assembly of bacterial communities is an indication of a potential functional change driven by environmental and soil chemical dynamics. For this study, RDA analysis revealed that phosphorus, potassium and pH were the soil chemical parameters that influenced the assembly of bacterial communities. Koshlaf et al.  reported that the addition of nutrients to an oil-polluted soil led to a significant change in the bacterial community structure after 2–4 months of remediation while Obieze et al.  demonstrated that potassium, pH and phosphorus were among important chemical factors that influence the assemblages of bacterial communities during hydrocarbon remediation. The significant correlation of these soil chemical parameters with the centroid of the samples drawn during remediation implies that their availability can significantly affect the recovery rate of artisanal refining sites.
Cyanobacteria and Proteobacteria were differentially (FDR-adjusted p < 0.05) abundant during remediation while Hydrogenedentes, Spirochaetes, Armatimonadetes, Caldiserica, Cloacimonetes and Deferribacteres were differentially abundant pre-remediation. Proteobacteria are copiotrophs thus their significant increase during remediation is in response to the addition of nutrients to the oil-polluted soil. Also, several species of this phylum are established hydrocarbon degraders [7, 45] and may have increased in relative abundance due to more favourable environmental conditions during remediation. Meanwhile, Cyanobacteria are photosynthetic microorganisms that exist in diverse environments and are known to quickly adapt to fluctuating environmental nutrient conditions [46, 47]. They also promote soil stability and some species are capable of carbon and nitrogen fixation (Diazotrophic species) . For this study, their increase in relative abundance may have contributed to the increased availability of nitrogen for the multiplication of hydrocarbon-degrading microorganisms. At the genus taxonomic rank Syntrophus, Syntrophobacter, Smithella, Pelolinea, Methanosaeta, Leptolinea, Bryobacter, Desulfobacca and Caldisericus were differentially abundant pre-remediation. The detection of established syntrophic, methanogenic, sulfate-reducing and nitrogen metabolizing bacterial and archaeal species as biomarkers suggest that prior to remediation, methanogenic hydrocarbon degradation was one of the main routes for carbon sequestration. Methanogenic hydrocarbon degradation is an important process in the biogeochemical carbon cycle . Several reports have demonstrated that the mineralization of hydrocarbons through methanogenesis involves a syntrophic relationship beginning with the initial activation of hydrocarbon substrates and subsequent formation of intermediates for methane production [49–53]. The community is said to either comprise hydrogenotrophic methanogens (Candidatus Methanoregula, Methanolinea, Methanospirillum), methylotrophic methanogens (Methanolobus) and/or acetotrophic methanogens (Methanosaeta) while the hydrocarbon activators usually include Deltaproteobacteria (Syntrophus, Smithella, Desulfovibrio, Geobacter) or other Proteobacteria, Verrucomicrobia or Firmicutes bacterial groups [54, 55]. Similar to this study, Liang et al.  detected Methanosaeta among the dominant archaea in an alkane-dependent methanogenic culture while Smithella was recently reported to be responsible for the oxidation of long-chain alkanes (C16 – C20) through fumarate addition during methanogenic hydrocarbon degradation .
PICRUSt2 predicted pathways revealed that there were significant differences in the microbial community function pre-remediation compared to the samples obtained during remediation. The differences in function corroborate the differences in the core microbiome pre-remediation and during remediation. Among pathways differentially abundant pre-remediation were those associated with methanogenesis and the biosynthesis of terpenoids. The detection of pathways for the biosynthesis of terpenoids indicates that methanogenic archaeal species were major contributors to the microbial community function as terpenoids are major components of methanogenic bacterial cells membrane [57, 58]. Meanwhile, the detection of several pathways associated with methanogenesis pre-remediation suggests that decades of artisanal refining in the Niger Delta can transform the site into a reservoir for methanogenic hydrocarbon degradation. The dominating presence of acetoclastic methanogens (Methanosaeta) and syntrophic hydrocarbon activating bacterial species (Smithella, Synthrophus) pre-remediation further establishes this fact. Roy et al.  had earlier suggested that under anoxic conditions, the activation of hydrocarbons and subsequent production of intermediates such as acetate, formate, hydrogen and methanol can trigger methanogenic hydrocarbon degradation. Furthermore, in sites where hydrocarbons make up a greater proportion of the organic matter, methanogenic hydrocarbon degradation may become the main carbon sequestration route . Keeping this in mind together with our findings, we conclude that the characteristic low energy yield associated with hydrocarbon degradation under anoxic conditions is one of the reasons for the prolonged natural recovery of crude oil polluted artisanal refining sites in the Niger Delta.