| Land oil spills are global extensive environmental challenges in oil-producing countries,including Nigeria,which has a long history of contaminated soils,groundwater,and streams by spill extension being the primary of numerous ordeals since oil exploration in 1958.At over2.5 million barrels of daily oil production,Nigeria is the highest oil-producing country in Africa and the sixth globally.In Nigeria,through petroleum storage and transportation accidents,operational failures,and illegal refining sites,oil spills have become intractably prevalent in the oil-producing region of Nigeria,known as the Niger Delta region.These have left the Niger Delta region of Nigeria harboring the oilfields and pipelines in crucial need of soil remediation to restore their agricultural and social-economic livelihood.To a considerable extent,several remediation attempts to restore previously contaminated sites have recorded relative successes.However,flawed remediation approaches in most areas without explicit knowledge of hydrocarbon utilizing microbial communities present have left patches of residual crude oil still lingering on soil surfaces and beneath.The United Nations Environmental Protection(UNEP)agency,in an earlier investigation on spill sites,recorded that 10 out of every 15 attempted clean-up and closed sites still showed patches of oil contamination and vertical oil migration into groundwater,which were still above the Shell Petroleum Development Company(SPDC),and Nigerian government remediation closure values of 50 mg kg-1.The analysis of spill data provided by the SPDC revealed that a total of 44 incidents of land oil spills?500 bbl amounted to 53,631 bbl between 2011 and 2019,which were primarily attributed to 83%sabotage.About 73%of the 53,631 bbl spilled crude oil were unrecovered from the spill areas,which had deleterious impacts on farmlands,fishponds,rivers,residential areas,and groundwater.Remediation by enhanced natural attenuation(RENA)is a common feasible landfarming technique for restoring the petroleum hydrocarbon-contaminated sites in Nigeria.The RENA approaches include non-supplemented and supplemented remediation using fertilizer,agricultural biomass,and animal wastes,which could be ineffective in cases without a proper understanding of the soil microbial community structure,underlying environmental factors,and the nature of contaminant crude oil.Therefore,it is crucial to understand the microbial community structures in contaminated soils and microbial responses to petroleum contaminants from different oilfields.This study carried out five aspects of research work:(1)To assess the history and current situation of Nigeria's oil pollution and its remediation approaches;(2)To study the oil-contaminated soils of three different oil fields in Delta State Nigeria,including the analysis of soil physicochemical properties,microbial communities,and the petroleum component compounds;(3)Comparative analysis of the differences in the structure and succession of microbial communities in oil-contaminated soils between China and Nigeria;(4)The use of crude oil from the three oilfields in Delta State as the sole carbon source to test their amenability to bacteria strains isolated from the contaminated soils.The oil-contaminated soil bacteria from the three different oilfields were separately cultured and purified.The oil-degrading ability of bacteria from different sources was analyzed;(5)The relationship between microbial communities and cultured bacteria from different sources and the degradation of crude oil pollutants was discussed.The results of this study expand the understanding of the impact of oil pollution on soil microbial communities in Nigeria and its response to oil pollution and provide scientific data for the microbial remediation of oil pollution in Nigeria.1.To understand how petroleum contamination and soil physicochemical properties jointly shaped the bacterial and fungal communities'structure of soils from different oilfields,high-throughput sequencing of 16S r RNA gene and 18S r RNA gene ITS amplicons were used to evaluate the shifts of microbial communities in the petroleum-contaminated soils in Ughelli East(UE),Utorogu(UT),and Ughelli West(UW)oilfields located in Delta State,Nigeria.A total of 1,515 bacteria and 919 fungal average OTU numbers were obtained from the three soil samples.The microbial community richness and diversity trend as AL>UT>UW>UE and AL>UW>UT>UE for bacteria,and AL>UW>UT>UE and UW>UT>AL>UE for fungi,respectively.The results showed that oil pollution reduces the diversity of bacterial and fungal communities in the soil,and the diversity of bacterial and fungal communities in oil-contaminated soils in different oilfields is quite different.A total of 42 bacterial phyla and 360 bacterial families were obtained by sequencing analysis.Proteobacteria and Actinobacteria were the most ubiquitously abundant bacterial phyla predominant in both petroleum-contaminated and non-contaminated soils.In the contaminated soils,the predominant bacterial phyla account for 82%of the total community abundance,including Proteobacteria,Actinobacteria,Acidobacteria,and Chloroflexi by 44%,17%,12%,and 9%of the bacterial phylum.In contrast,in the UT soil,the abundance of Bacteroidetes,Firmicutes,Fusobacteria,and Planctomycetes was very high and twice that of UE and UW soil.In UT,UE,and UW soils,the bacteria families with the highest degree of dominance are unclassified Rhizobiales(14.4%),KCM-B-112(12.8%),and Acidobacteriaceae(8.5%),while other dominant bacteria families are Desulfurellaceae,Rhodospirillaceae,and Anaerolineaceae.Comparatively,the abundances of Proteobacteria and Acidobacteria in the uncontaminated soil were significantly reduced to 27.2%and 8.4%,respectively.The result shows that the dominant groups of soil bacteria and the community structure they represent are different in various oil-contaminated soils,relative to the differences in soil environmental factors and petroleum components.A total of 8 fungal phyla,196 fungal families,and 419 fungal genera were obtained by sequencing analysis.Ascomycota,unclassified Fungi,and Basidiomycota represented 50.3%,42.2%,and 6.2%of fungal phylum.The abundance of Ascomycota was 2.3%in AL soil,but its abundance in oil-contaminated soil UE,UT,and UW were 41.5%,55.9%,and 54.2%,respectively.Unclassified Fungi phylum was most dominant in UE soil but relatively reduced in UT and UW soils.Compared with AL soil,the abundance of Chaetomiaceae,Nectriaceae,unclassified Hypocreales,and Sporormiaceae in petroleum-contaminated soils was significantly reduced.It is worth noting that the fungal families unclassified Sporidiobolales and Montagnulaceae,which are related to petroleum degradation,account for 10.6%and 5.2%of UT,respectively.Unclassified Chaetothyriales and Pleosporaceae account for 7%and 6%of UW,and unclassified Hypocreales account for 5%of UE.Aspergillus dominates in uncontaminated AL soil at the genus level,while unclassified Fungi dominates in contaminated soil.The dominance of Aspergillus,Talaromyces,unclassified Sporidiobolales,and Paraconiothyrium in UT is significantly higher than UW and UE.These results indicate that oil contamination has changed the soil fungal community structure,and the dominant groups of oil-contaminated soil are also different in various oilfields.Principal coordinate analysis(PCo A)results show that the bacterial and fungal communities of AL soil and the three petroleum-contaminated soils are significantly different,and the bacterial and fungal communities of the three petroleum-contaminated soils also have distinct differences,indicating that the microbial communities and distribution areas and petroleum components are different.The Redundancy analysis(RDA)and Spearman correlation show that total the soil N(TN),carbon(TC)and moisture content(MO)showed a significant positive correlation to Alpha-,Delta-,Beta-,Gamma-proteobacteria,Acidobacteria,Anaerolineae,and unclassified Saccharibacteria,while soil electrical conductivity(EC)and p H were significantly positively correlated with Actinobacteria,Thermomicrobia,and KD4-96,indicating the synergistic effect of these soil factors and oil pollution on these bacterial communities.For fungal communities,MO,TN,and TC were positively correlated with unclassified Sporidiobolales,Pleosporaceae,Montagnulaceae,and unclassified Fungi,while EC was positively correlated with Trichomaceae,Chaetomiaceae,and Nectriaceae.There was no significant correlation between soil p H and fungal genera.Among the contaminated soils,the highest soil organic matter(SOM)was 13.72%in UE,4.55%in UW,and 3.64%in uncontaminated AL,indicating significant differences between soil samples.In all contaminated soil samples,there was a negative correlation between SOM and OTU-based bacterial community abundance and diversity and a significant positive correlation with fungal community abundance(p<0.05),which could be attributed to its effect on substrate bioavailability.Further Spearman correlation analysis revealed the differences in bacterial and fungal communities'responses to soil physicochemical factors.Over 30%of bacterial genera statistically correlated positively or negatively with soil p H,while none in the fungal genera.Conversely,only unclassified Rhizobiales positively correlated with soil MO in the bacterial genera,while 20%of fungal genera statistically correlated positively with soil MO,which indicated that fungi have a higher tolerance for the narrow soil p H range but most sensitive to soil moisture compared to bacteria.Notably,unclassified Acidobacteria and unclassified Acidimicrobiales most statistically correlated p H and EC by 47%and 43%,respectively.Anearolinea was most negatively correlated with EC but positively correlated with TC,TN,and MO by 53%,50%,and 43%,respectively,while unclassified Rhizobiales were positively correlated with MO.The fungal community most positively correlated with MO,TN,and TC by 40%,33%,and 40%,respectively,notably Paraconiothyrium and Pyrenochaetopsis genera,while 57%of the fungal genera were positively correlated with EC.The bacterial taxa KCM-B-112,unclassified Saccharibacteria,unclassified Rhizobiales,Desulfurellaceae,and Acidobacteriaceae and fungal Trichocomaceae,unclassified Ascomycota,unclassified Sporidiobolales,and unclassified Fungi were found to be the dominant families in petroleum-contaminated soils.These dominant families are well known to contain hydrocarbon utilizing bacterial and fungal genera,which is also reflected in the principal coordinate analysis(PCo A)visual evaluation of microbial community responses to soil petroleum contamination and physicochemical properties.The correlation between bacterial and fungal community relationship to soil physicochemical properties using the Redundancy analysis(RDA)and Spearman correlation analysis revealed that total carbon(TC),electric conductivity(EC),p H,SOM,and moisture content(MO)were the significant drivers of bacterial and fungal communities,respectively.Gas chromatography-mass spectrometry(GC-MS)was used to analyze the petroleum components from three sources.The analysis revealed that low molecular weight(LMW)compounds in UT contaminant crude oil include cyclohexane,ethylbenzene,p-xylene,benzene,1H-indene,and m-menthane,which differ from UE and UW crude oil with higher contents of mid molecular weight(MMW)and high molecular weight(HMW)compounds and are partly responsible for the significant disproportion in some vital bacterial and fungal phylotypes.Significant differences in component composition ranging between C7–C10,C11–C16,and C12–C29 compounds abundance in respective crude oils and soil MO content in UT contaminated soil jointly contributed to the microbial community variance.However,the UE crude oil had the highest content of both MMW and HMW compounds.Phylogenetic Investigation of Communities by Reconstruction of Unobserved States(PICRUSt)software was used to predict and analyze the bacterial community function,and the predicted protein was classified as Kyoto Encyclopedia of Genes and Genomes(KEGG)orthologs(KOs).A total of 319 pathways were predicted;the most abundant pathways include Ribosome,Aminoacyl-t RNA biosynthesis,Citrate cycle(TCA cycle),NADH:quinone oxidoreductase,and Branched-chain amino acid transport system.Compared with uncontaminated soil,the high abundance pathways in petroleum-contaminated soil include Benzoyl-Co A degradation,Methanogenesis,Methane oxidation,methanotroph,Cymene degradation,and Naphthalene Degradation.The abundance of RNA polymerase,Methionine biosynthesis,Pantothenate biosynthesis,Vancomycin resistance,D-Ala-D-Lac type,and Che A-Che YBV(chemotaxis)two-component regulatory system increased significantly in uncontaminated soil.In the three contaminated soils,most hydrocarbon degradation pathways have their highest abundance in UE and the lowest in UT,which shows a higher contamination level with non-volatile and recalcitrant petroleum compounds.Kos related to petroleum hydrocarbon degradation in petroleum-contaminated soil includes Benzoate degradation(Ko00362),Xylene degradation(Ko00622),Lysine degradation(Ko00310),Tryptophan metabolism(Ko00380),Phenylalanine,tyrosine and tryptophan biosynthesis(Ko00400),Naphthalene degradation(KO00626),Propanoate metabolism(Ko00640),Methane metabolism(Ko00680),Nitrogen metabolism(Ko00910),Fatty acid metabolism(Ko00071)and degradation of aromatic compounds(Ko001220).In uncontaminated soil,the core iconic Ko is Ascorbate and aldarate metabolism(Ko03021),Cysteine and methionine metabolism(Ko00270),Valine,leucine,and isoleucine biosynthesis(Ko00290),Lysine biosynthesis(Ko00300),Pantothenate and Co A biosynthesis(Ko00770),Flavonoid system(Ko00770)941,Two-component),and Bacterial chemotaxis(Ko02030).These results show that there are diverse and abundant petroleum hydrocarbon degradation pathways in petroleum-contaminated soil,including methyl benzoate xylene degradation(M00537),catechol toluene degradation(M00538),benzoate/benzoyl-Co A degradation(M00540-1 and M00551),and many other pathways involved in aromatic degradation,as well as sulfate reduction(M00176),dissimilative sulfate reduction(M00596)and sulfate transport system(M00185)These metabolic characteristics indicate that the microbial community depends on polluting petroleum hydrocarbons as the sole carbon source.This predictive profiling revealed the high presence of functional degradation pathways indicating the increased presence of bacteria harboring genes responsible for various hydrocarbon degradation.The study revealed bacterial and fungal communities responsible for the biodegradation of petroleum contamination in these oilfields,which could serve as biomarkers to monitor contaminated soil restoration within these areas.Further studies on other contributing factors such as heavy metal content in these contaminated sites are recommended,offering valuable insights into oil spill impact on microbial communities.2.Comparative analysis of the microbial community structure of oil-contaminated soil in China and Nigeria oilfieldsChina and Nigeria presently rank fourth and fifteenth largest oil-producing countries globally,making oil spill contamination a commonly shared environmental issue of concern between both countries.Like Nigeria,China has recorded a significant magnitude of oil spills in coastal areas and land.The impact of oil-polluted soils contributed to the 2016 Action Plan on Preventing and Controlling Soil Pollution in China centered on remediation of contaminated soils.This plan is estimated to achieve 90%reclamation and utilization of contaminated agricultural land sites by 2020 and 95%by 2030.A comparative analysis of predominant microbial communities in oil-contaminated remediating sites between China and Nigeria could better understand the differences and similarities in microbial response to petroleum contamination.It is also crucial to understand the dynamics of environmental factors affecting microbial communities,which could be used as biomarkers to bioremediation of oil spill environments.Besides soil petroleum contamination are also environmental factors relative to regional and geological differences,which play a significant role in soil microbial community structure.China has?400 oilfields distributed in 25 provinces,including autonomous regions across China,unlike Nigeria,where?159 oilfields onshore are distributed within the Niger Delta region.The predominant phyla shared between both country's contaminated soils include Proteobacteria,Actinobacteria,Acidobacteria,Chloroflexi,Bacteroidetes,Planctomycetes,Verrucomicrobia,Saccharibacteria,Cyanobacteria,Chlorobi bacteria,and Crenarchaeota(Archaea)bacterial,Ascomycota,Basidiomycota,Chytridiomycota,Zygomycota,and Glomeromycota fungal.Among the predominant phyla,Actinobacteria and Cyanobacteria have a higher abundance in non-contaminated soils,attributable to the ubiquity and opportunist degradation capability common in petroleum-contaminated soils in Nigeria and China.The most frequently occurring genera shared in both country's contaminated soils were Pseudomonas,Rhodococcus,Bacillus Aspergillus,and Penicillium at varying degrees of abundance across contaminated oilfield soils.Some studies in aged-contaminated soils in China reported a positive correlation between petroleum contamination and microbial diversity;conversely,most of Nigeria's studies reported a negative correlation with microbial diversity.However,petroleum contamination age was a significant contributing factor determining their adaptation period for specialized mineralization,notable among contaminated oilfield soils between the 1960s,1970s,and 1990s.Microbial metabolisms for hydrocarbons are most specific at the species level,so purified single oil-degrading strains produce fewer enzymes and lower concentrations for specific compounds,suggesting that a consortium of mixed strains could be better degraders than a single strain—conversely,some distinct strains like Pseudomonas sp.ZS1 was found to have an inhibitory effect on Donghicola sp.CT5 and Bacillus sp.CT6,while Alcaligenes sp.CT10 had an antagonistic effect on Pseudomonas sp.ZS1.The influential factors of microbial abundance differences include increased aromaticity,contaminant concentration,and soil physicochemical properties such as soil type,temperature,soil p H,moisture content,and organic and inorganic supplement.3.Study on the isolation and characterization of bacteria degrading strains from oil-contaminated soils in three oilfields in Delta State,Nigeria.The bioremediation of petroleum pollutants in the environment relies on the complete mineralization of component compounds by single axenic strains or consortia of mixed strains.Isolation,characterization,and profiling of strains with unique degradation capability are crucial in selecting viable petroleum degrading strains.Several hydrocarbon degradation capabilities tests have evolved several screening techniques,such as microtiter turbidity,miniature sheen screens,oxygen consumption,and carbon dioxide release.However,these techniques are either impeded by time and are labor-intensive,unreliable results,or cost-ineffective.Thus,a colorimetric method using DCPIP redox indicator is one of the strategies for a precise assessment of microbial biodegradation capability,which is proven to be quick,simple,and low-cost effective.Thirty-seven bacterial strains were isolated from crude oil contaminated soils from three different oilfields in Delta State,namely Utorogu(UT),Ugheli East(UE),and Ugheli West(UW)oilfields.The instant qualitative DCPIP degradation by colorimetric assessment revealed varying degradation capabilities by color gradient:C-Blue,B-Light blue,and A-colorless,representing weak,moderate,and strong degrader,respectively.The qualitative degradation results were UE strains(%population):2"C"(16.6%),2"B"(16.6%),and 8"A"(66.6%),UT strains:2"C"(16.6%),2"B"(16.6%),8"A"(66.6%),and UW strains:3"C"(25%),1"B"(8.3%),and 7"A"(58.3%)degradation capacity.Further degradation assessment by UV spectrophotometer absorbance at 600 nm in 96 hours was used as a quantitative assessment criterion for strain selection.However,all the strains showed a consistent increasing degradation,but eight strains with the most degradation potentials were selected for identification and further degradation assay.Selected strains were identified by PCR gene analysis and characterized as Ochrobactrum intermedium strain CC15(E2),Ochrobactrum intermedium strain XG-2(E6),and Pseudomonas stutzeri strain S3(E3)from UE oilfield,Stenotrophomonas maltophilia strain TS51(T3),Lysinibacillus fusiformis strain Rizhao?587?1(T6),and Lysinibacillus sphaericus strain ARg(T7)from UT oilfield,Bacillus cereus strain S5(W11),and Ochrobactrum strain ES-QY-2(W8)from UW oilfield.The selected strains in axenic and consortium cultures were subjected to their respective oilfields'crude oil degradation for 35 days at intervals of 7,20,and 35 sampling time for gravimetric analysis,cell growth optical density,and CFU assessment.The results revealed that Lysinibacillus fusiformis strain Rizhao?587?1(T6)23.55%,Pseudomonas stutzeri strain S3(E3)44.5%,and Bacillus cereus strain S5(W11)at 44%degradation were the best degrading strains from respective oilfields.The UT crude oil had the highest amenability to the consortium culture of mixed strains(Tmix)by a synergetic effect.The UW and UE crude oil showed lesser amenability to the consortium culture of mixed strains Umix and Wmix culture,which suggested a weaker synergy in degradation.The regression analysis shows that the optical cell growth density and CFU positively correlated with crude oil degradation.GC-MS was used to analyze the residual UT crude oil to determine compound utilization by the isolated strains Stenotrophomonas maltophilia TS51(T3),Lysinibacillus fusiformis Rizhao 587-1(T6),and Lysinibacillus sphaericus ARg and the mixed cultures.When comparing the cultured media with the uninoculated medium,the result showed that in the petroleum residue after strain degradation,low-molecular-weight(LMW)alkanes from C7H14to C10H20 were not detected,indicating that this part of the hydrocarbons was completely degraded and utilized by the strains.Mid-molecular weight(MMW)compound typical peaks,such as 1-Nonylcycloheptane C16H32,1-Decanol,2-hexyl-C16H34O,and high molecular weight(HMW)compound typical peaks,such as Oxalic acid and heptadecyl C29H54O4,were notably absent from the MS spectrum.The disappearance of these hydrocarbon compounds indicates that the strains can also degrade and utilize these petroleum hydrocarbon compounds.Degradation similarities and differences across the three axenic stains were the recalcitrant compounds such as Pulegone C10H16O,4a,10a-Methanophenanthren-9.beta.-ol C15H17Br O,Benz[e]azulen-3(3a H)-one C17H24O5,1H-2,8a-Methanocyclopenta[a]cyclopropa C20H28O6,Dasycarpidan-1-methanol,acetate(ester),4H-Cyclopropa[5',6']benz[1',2':7,8]azuleno C22H30O8,which were unamenable.In contrast,the Tmix culture synergy completely utilized Pulegone C10H16O,4H-Cyclopropa[5',6']benz[1',2':7,8]azuleno C22H30O8,and 5H-Cyclopropa[3,4]benz[1,2-e]azulen C22H30O7,compounds,unlike any of the single strains.Importantly,while some heavy compounds were broken down,new compounds originally not found in the control were formed including 5H-Cyclopropa[3,4]benz[1,2-e]azulen C22H30O7,2(1H)-Naphthalenone,octahydro-8a-methyl C11H18O,Phenol,2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl C23H32O2,2,6,10,14,18,22-Tetracosahexaene C30H48,and Cyclotrisiloxane,hexamethyl-C6H18O3Si3while Pulegone C10H16O,Dasycarpidan-1-methanol,acetate(ester)C20H26N2O2 and 4H-Cyclopropa[5',6']benz[1',2':7,8]azuleno C22H30O8 peaks occurrence was increased by>100%.The three strains of Ochrobactrum intermedium CC15(E2),Pseudomonas stutzeri S3(E3),and Ochrobactrum intermedium SG2(E6)isolated from the UE oil field have great differences in hydrocarbon utilization.Among these compounds,C11H18O to C14H24O and HMW 2-[4-methyl-6-(2,6,6-trimethylcyclohex C23H32O compound were completely used up,but most HMW were recalcitrant.Although similar compounds were amenable to Ochrobactrum intermedium strain SG2(E6)and Pseudomonas stutzeri strain S3(E3)and Ochrobactrum intermedium strain CC15(E2).In contrast,Ochrobactrum intermedium strain SG2(E6)had the highest reduction of HMW 4H-Cyclopropa[5',6']benz[1',2':7,8]C22H30O8and1H-2,8a-Methanocyclopenta[a]cyclopropa C20H28O6 compounds by 77%and 93%peaks,respectively,compared to the control.Conversely,Emix culture did not significantly form the expected stronger synergy to utilize more hydrocarbon compounds when compared to the single strains.Like other cultures,Emix utilized lower MMW compounds but had the least reduction of 4H-Cyclopropa[5',6']benz[1',2':7,8]C22H30O8 by 46%.However,some new compounds formed include Cyclotrisiloxane,hexamethyl C6H18O3Si3,Phenol,2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl C23H32O2.8,14-Seco-3,19-epoxyandrostane-8,14-dione,17-acetoxy-3.beta.C24H36O6,and Androst-5,7-dien-3-ol-17-one C19H26O2.The UW crude oil residue revealed that Ochrobactrum ES-QY-2(W8)and Bacillus cereus S5(W11)isolated from UW oilfield and their mixed culture could completely utilize MMW compounds C10H16O to C14H24O.Ochrobactrum ES-QY-2(W8)completely utilized some HMW compounds such as Phorbol C20H28O6 and Fenretinide C26H33NO,while Bacillus cereus S5(W11)utilized 69.75%4H-Cyclopropa[5',6']benz[1',2':7,8]Azuleno C22H30O8 and 6-Methyl-11-propenyl-5-(toluene-4-sulfonyloxy)C24H32O7S,the mixed culture of the two strains also did not show a positive synergy.However,The Wmix culture of all the isolated strains did not create a significant degradation synergy for hydrocarbon compound utilization.In summary,C7-C10 and C16 low molecular weight(LMW)compounds in UT crude oil,C11-C15 and C23 medium molecular weight(MMW)compounds in UE crude oil,and C10-C14compounds in UW crude oil were most amenable to bacterial strains,while High molecular weight(HMW)compounds C16-C37 are the most recalcitrant compounds.The differences in the strains'ability to utilize various MMW and HMW compounds indicate that the bacteria isolated from the contaminated soils in different oilfields are also related to the composition of petroleum hydrocarbons in their living environment.The mixed strain culture did not have a positive synergistic effect on the degradation of most complex HMW compounds. |
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