Selection and quantification of key functional genes of some selected bacterial species in a microcosms study for biodegradation of crude oil

doi:10.21203/rs.3.rs-4806599/v1

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Selection and quantification of key functional genes of some selected bacterial species in a microcosms study for biodegradation of crude oil

https://doi.org/10.21203/rs.3.rs-4806599/v1

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GC-MS and qPCR have been used to facilitate the profiling of metabolites from a wide range of oil materials leading to the wide coverage of comprehensive central pathways involving primary metabolism and the quantification of functional genes responsible for the biodegradation of crude oil components. Therefore, the present study aimed to explore the ability of Pseudomonas aeruginosa and Pseudomonas lurida for the biodegradation of crude oil. The results of the GC-MS analysis showed an extensive elimination of hydrocarbons of mostly low and medium-chain hydrocarbons. The qPCR analysis was carried out to determine the activity of the functional genes and showed a substantially higher relative fold expression of 4-hydroxybenzoate monooxygenase gene (Ben) of 2.1x1014 fold after the first week (T1) during the biodegradation study with P. lurida. However, low relative gene fold of 60.91 for catechol-2,3-dioxtgenase gene (cat23) was observed. In the same vein, the relative fold expression of 2156.87 was detected for alkane monooxygenase (alkB) gene from a study with P. aeruginosa. This is substantially higher than the expression for cat23 gene and greatly lower than the Ben gene. The overall results of this study could evidently prove the environmental application of these bacterial species – B. endophyticus P. aeruginosa and P. lurida for the management of crude oil polluted environments. Hence, the overall finding from this study could be utilised as a tool to design an engineered bioremediation process to address the long devastating crude oil pollution across the Niger Delta and beyond.

In Nigeria, Shell D’Arcy began the search for crude oil in the late 1930s at Iho in the North West province of Owerri. The first commercially viable oilfield was discovered at Oloibiri, a small town in Bayelsa State in the Niger Delta in 1956 (Odinioha and Brisibe, 2013). Subsequent exploration for crude oil by leading companies has grown resulting in the production of Bonny light crude oil in the region (Okoh, 2003). More than 80% of Nigeria’s crude oil originated from the offshore and onshore parts of the Niger Delta (Chikere et al. 2009). Generally, crude oil pollution has been a great challenge till today, resulting from activities of the related industries. Crude oil spills in both aquatic and terrestrial surroundings have the potential to cause significant impacts on soil fertility, vegetation, aquatic organisms, and microbial community structures (Kgopa et al., 2018; Kadafa, 2013; Tolulope, 2004).

One of the best approaches to monitor the biodegradation of crude oil is through the use of gas chromatography-mass spectrometry (GC-MS) and quantitative polymerase chain reaction (qPCR) instruments. Through GC-MS-based studies, it is possible to gain insight into the rate at which biodegradation is occurring in the field or the laboratory (Alnuaimi et al. 2020). Likewise, the technique has the potential to facilitate a robust quantification and enhanced identification of the composition of the crude oil. This analytical tool has been used to facilitate the profiling of metabolites from a wide range of oil materials leading to the wide coverage of comprehensive central pathways involving primary metabolism (Lisec et al., 2015). Hence, this is what makes the technique more suitable for monitoring the biodegradation of crude oil (Alnuaimi et al. 2020).

Previous studies in Nigeria and elsewhere have indicated the efficiency of several bacterial species in degrading a wide range of hydrocarbons including crude oil. For instance, Nkiru et al. (2019) investigated the total petroleum hydrocarbon in a broth culture study using bacterial species isolated from three different locations in the Niger Delta region. Their finding failed to report what kind of hydrocarbons were eliminated or was lowered by the six bacterial species used in their study. Also, a study by Abdullah et al., (2019) documented the ability of some bacterial species to degrade a wide range of hydrocarbons, as indicated by the substantial changes in the composition of hydrocarbons. Alnuaimi et al. (2020) used GC-MS to evaluate the effectiveness of Bacillus megaterium in biodegrading crude oil in the Baghdad area of Iraq. Hence, proving the effectiveness of the bacterial species in mineralizing the components of crude oil. Chen et al., (2017) highlighted the efficiency of five bacterial strains Pseudomonas aeruginosa ASW-2, Alcaligenes sp. ASS-1, Exiguobacterium sp. ASW-1, Alcaligenes sp. ASW-3 and Bacillus sp. ASS-2 in degrading 75% of Chinese crude oil in 7 days. In this study, GC-MS data displayed a gradual increase in the efficiency of biodegradation of C13 to C16 n-alkanes from 41.3–60.7%, while a stagnant degradation efficiency of close to 44% was observed in the C17 to C24. However, a higher degradation efficiency was noted among alkanes C25 to C30 n-alkanes which increased from 60.2–67.9%, and lastly, an increased degradation efficiency of the free bacterial consortium was noted to have increased among the alkanes of C30 to C32 n-alkanes between the values from 60.2–79.2%.

Furthermore, bacteria that have the exceptional ability to mobilize a wide range of hydrocarbons are commonly found in the environment. These bacterial species have been characterized with a dynamic change based on the community structure, depending on their different abilities to mineralize crude oil. The impact of the indigenous microbial consortium has been well established by Asadirad et al., (2016), in a microcosm study. This study, which used Bacillus subtilis and Bacillus licheniformis confirmed the degradation of petroleum hydrocarbon by up to 77.5% during the first 10 days of this study. The study concludes that the highest depletion of crude oil was observed during these first 10 days with 77.5% being the 166 highest biodegradation level. Gao et al. (2016) exploited an exogenous B. subtilis to improve the process of indigenous microbial enhanced oil recovery (IMEOR). The core-flooding test indicated an increased efficiency of oil displacement of 16.71% by both indigenous and exogenous against the 7.59% noted in the control which had nutrients.

It has been observed that the native bacterial species that harbour alkane catabolic genes have the ability for the biodegradation of crude oil-polluted soil and sediment (Liu et al., 2014; Paisse et al. 2013; Kloos et al. 2006). Alkane monooxygenase (alkB) gene is the major gene critical during the first step of hydrolysis for the metabolism of alkanes (Liu et al. 2014; Beilen et al. 2007). For instance, a study has revealed the critical role of alkane monooxygenase enzymes in the bacterial degradation routes involving alkanes (Whyte et al. 2002). However, few bacterial species have been characterized for this critical gene in a microcosms study. For example, Pseudomonas oleovorans and Pseudomonas putida were reported to have utilized C5 – C12 n-alkane in a study that examined the alkB gene (Beilen et al. 1994; Beilen et al. 2001). Likewise, another study indicated the utilization of C10 – C20 n-alkanes by Acinetobacter sp. where the alkM gene was well characterized (Ratajczak et al., 1998). More recently, Agbaji et al., (2021), examined the catabolic versatility of 1,2-alkane monooxygenase, catechol 2,3-dioxygenase, and naphthalene oxygenase genes during a biodegradation study involving aromatics and n-alkanes by Achromobacter agalis, Pseudomonas fluorescens, Staphylococcus lentus, and Bacillus thuringiensis. For this study, the bacterial species were found to have utilized C₈ – C₁₇, C₁₈ – C₃₀, and phytane.

Real-time PCR is an accurate and very sensitive technique that permits the quantification of functional genes coding the enzymes degrading the components of crude oil through the detection of PCR product in real-time (Shahsavari et al., 2016). Due to the crucial implication of enzymes such as dioxygenases and monooxygenase during the biodegradation of crude oil, the present study aimed to implore the application of both GC-MS and qPCR for the biodegradation of crude oil thereby examine the expression level of key functional genes and quantification of metabolites being degraded by a novel (P. lurida) and common (P. aeruginosa) bacterial species.

Sample collection and processing for the biodegradation of crude oil

The bonny light crude oil used for this study originated from the Niger Delta region and was obtained from the department of microbiology Bayero University Kano, Nigeria. The crude oil sample was stored in a safety cabinet upon arrival at the University of Greenwich at Medway. The sediment samples used for the study of the microcosm were collected from Medway River during the low tide and transported to the molecular biology research laboratory. Sediment samples were preserved in the fridge at 4°C until the commencement of the experiment. All the sediment samples used in this study were autoclaved repeatedly until there were no viable bacterial cells on agar plates.

Preparation of the experimental setup

For this study, Pseudomonas aeruginosa (NCTC 10662) was purchased from the National Collection of Type of Cultures (NCTC), Bacillus subtilis (B8A160056) were purchased from Philip Harris (UK) whereas both Pseudomonas lurida (DSM15835) and Bacillus endophyticus (DSM13796) were purchased from DMSZ Leibniz Institute, Germany. P. aeruginosa was supplied in solid form and a viable pure culture of P. lurida, B. subtilis, and B. endophyticus was inoculated individually in a sterile 250 ml flask containing fresh LB broth (ThermoFisher Scientific) prepared according to manufacturer’s instructions. The flasks were incubated in a shaker at 170 rpm at 37°C for 24 hours. Phosphate Buffer Saline (PBS) solution was prepared by dissolving one tablet per 200 ml of deionized water using a magnetic stirrer. Likewise, all autoclaving was performed for 15 minutes at 121°C. Bacterial cells were harvested by pipetting 1 ml of culture in sterile Eppendorf tubes and were centrifuged at 1000 rpm at 4°C for 5 minutes. After that, the supernatant was discarded, and the pallets were washed using sterile ice-cold PBS solution. This is done by adding 1 ml of sterile ice-cold PBS in the Eppendorf tubes containing the pallets and the tubes were centrifuged at 1000 rpm at 4°C for 5 minutes. This was carried out three times to ensure no nutrients from the medium were left behind. After that, all pallets were suspended in 1 ml of minimal salt medium (MSM). Thereafter, serial dilution was carried out to determine the bacterial colony-forming unit (CFU) per ml for the study of the microcosm. This was done by adding 1 ml of bacterial cells suspended in MSM into autoclaved tubes containing 9 ml of deionized water. Standard plate counting was performed by pipetting 100 µl from each serial dilution tubes into LB agar (ThermoFisher Scientific) plates prepared according to manufacturer’s instruction. All plates were incubated at 37°C for 24 hours.

Microcosms set up

For this study, 30g of autoclaved soil sample were added in sterile Petri dishes with 10% crude oil sterilized by passing through a 0.2µm filter. Thereafter, 5x10⁷ Colony Forming Unit of P. aeruginosa, and P. lurida, were inoculated in the microcosm runs in three biological replicates. The sediment sample was used as a negative control was not inoculated with any bacterial cells. All microcosms were carried out in three biological replicates for each bacterial species and mixed consortia. Day one (T0) samples were taken for GC-MS analysis after which the plates were incubated at 30°C. Samples for the GC-MS and functional gene analysis were taken after every seven days through the period of five weeks. Microcosms with P. aeruginosa and P. lurida were performed for the period of five and four weeks, respectively. Thus, the RNA templates used for the determination of gene expression in the selected microcosms were extracted using Total RNase Qiagen.

Sample Preparation and Gas Chromatography-Mass Spectrometry Analysis

For this study, 100 µl of each of the hydrocarbon standards (n-Cyclopantane, n-Hexane, n-Octane, n-Nonane, n-Decane, n-Undecane, n-Dodecane and then Tetradecane) and 200 µl of dichloromethane (DMC) were added in a tube to make 1mL in total. After that, 10 µl of the hydrocarbon standard mixture was added to a GC-MS vial containing 990 µl of dichloromethane to give a concentration of 10 µl per mL. This was repeated to give to 20 µl per mL, up to 50 µl per mL. After that, the GC-MS vials were sonicated for about 10 minutes. After which that, 1.0 µl was injected into the GC-MS for calibration using the same program/settings as the experimental samples described below.

Crude oil-polluted sediment (2g) from each of the microcosms was combined with 12 mL of dichloromethane and the mixture was sonicated for 3 minutes. The extract was centrifuged and filtered through a cotton cool plug contained within a sterile glass pipette. Sodium sulphate was used to remove moisture from the extracted mixture. This was done by adding the sodium sulphate in the Falcon tube containing the mixture and was shaken vigorously. After that, 2 µl of each microcosm sample was injected into a GC containing an HP-5MS column (Agilent) for the analysis of crude oil degradation by these bacteria. A temperature of 280°C was used for the injector while a temperature of 300°C was also used for the detector. Similarly, a holding temperature for 2 minutes at 4°C and 45°C to 310°C per minute as well as at 310° C for 25 minutes were maintained throughout the GC-MS runs. This analysis and the identification of the hydrocarbon compounds was done using CHEMSTATION software as well as NIST 2012 WILEY 2009 using default settings.

Real-time PCR experiment

In this procedure, each reaction was prepared with SYBR Green 10 µl, forward primers 0.4 µl, and reverse primers 0.4 µl, nuclease-free water 4.2 µl and then cDNA 5 µl. The qPCR cycling conditions in this study are initial denaturation step at 95°C for 2 min, 40 cycles of denaturation at 95°C for 15 sec, annealing temperature differs with each primer and lastly the extension period of 72°C for 30 sec. The signal acquisition was designed to occur during the extension period in order to avoid the detection of primer dimer rather than the right products. PowerUp™ SYBR™ Green Master Mix (Promega) used for the experiment contained an optimized buffer component, heat-liable UDG, ROX passive reference, 2x mix with SYBR Green dye, and Dual-lock Taq DNA polymerase (dNTPs which has dUTP/dTTP blend).

Analysis of qPCR data

The relative quantification method of analysis was carried out adopted by (Livak and Schmittgen, 2001) to determine the relative fold expression of the key functional genes. This method examined the relative change of gene expression. This was done by normalizing the Ct value of the target gene against the housekeeping gene (rRNA gene) using the formula (ΔCt = Ct target gene – Ct housekeeping gene). In this method of analysis, the ΔΔCt represent the difference of corrected Ct values among calibrator and the target gene. Finally, relative gene expression was determined using the 2-ΔΔCt formula. Likewise, the statistical analysis such as average Ct value and standard deviation were carried out using Excel software and all data analysed for qPCR analysis were presented as ± SEM. For this study, two of the three biological replicates of the microcosms were selected for functional gene analysis. Thus, a comparison was done between day one (T0) prior to incubation and week one (T1) following the incubation of all the microcosms.

For this study, the analysis of the GC-MS data was done using ChemStation software with two databases for mass spectral comparison. The primary database used was NIST version 2.0d Standard Reference Database. Analyte identification was done in ChemStation Enhanced Data Analysis by comparing spectra obtained from the GC-MS runs with spectra contained in the NIST SRD. Analyte matches with equal to or greater than 90% similarity to the NIST database.

The results of the GC-MS analysis obtained from the present study has shown an extensive elimination of a wide range of hydrocarbons by each of the bacterial species used for this study, presented in Fig. 1A–C and 2A–C. Most of these eliminated compounds are short-chain and medium-chain hydrocarbons. Similarly, a drastic change in the composition of the peak areas was also observed. Thus, suggesting partial biodegradation of the composition of crude oil. However, the GC-MS analysis indicated some other hydrocarbon compounds were unaffected throughout the duration of the microcosms study. This could suggests the inability of both P. aeruginosa and P. lurida to degrade these hydrocarbons compounds. Interestingly, the species were observed to have lowered the abundance of hydrocarbons used in this study.

GC-MS analysis of crude oil degradation by Pseudomonas aeruginosa

The chromatograms and bacterial growth curves in Figs. 1A, B, and C were obtained from a biodegradation study with a single strain of P. aeruginosa incubated at 30°C for a period of five weeks. The T0 chromatogram represents the crude oil profile on day one before being incubated while T5 showed the biodegradation profile of the components of crude in the fifth week when the experiment was terminated. The bacterial growth curve in Fig. 1C has indicated a rapid adaptation by Pseudomonas aeruginosa in the biodegradation study. The exponential growth by the bacterial species observed between T1 and T2 suggests a suitable utilization of the crude oil constituents by this bacterium. The bacterial growth was noted to have plateaued at T2 to T4 and then started declining suggesting that the preferential components were already being exhausted by bacterium (Fig. 1C). The GC analysis indicated an overall reduction of the abundance of the crude oil components by 50% in the soil microcosm with single strain P. aeruginosa. The GCMS showed the higher abundance of most carbon compounds present in the right side of the chromatogram at retention time between 40 and 52 (Fig. 1A and figure B). When compared the GCMS data in T0 (Fig. 1A) with T5 (Fig. 1B), it was observed that C₁₃ was broken down to C₆ at the same retention time i.e. broken down from Oxalic acid, allyl octadecyl ester to Butane, 2,2-dimethyl- (Table 1), C₁₂ to C₁₀, C₁₅ to C₁₄, C₂₁ to C₁₆ and another C₂₁ to C₂₀ (Table 1) all of which were partially degraded. Some hydrocarbon compounds were observed to have been degraded completely, and these compounds can be seen in the T0 GC chromatogram (Fig. 1A) e.g. C₆, C₇, C₉, C₁₆, C₁₇, and C₂₁ at retention time 6.88 min, 8.427 min, 43.219 min, 13.11 min, 37.279 min, 40.925 min, 46.755 min, respectively, but are not present at later in T5 GC chromatogram (Fig. 1B). A list of partially degraded and completely degraded carbon compounds is presented in Table 1. However, other compounds appeared to be the same throughout the process of biodegradation.

Table 1

List of degraded, partially degraded, and non-degraded compounds by *P. aerugenosa*
S/No	Compounds completely degraded	Compounds partially degraded	Compounds not degraded
1	O-xylene	Decane,4-methyl	Decahydro-4,4,8,9,10-pentamethylnaphthalene
2	Oxalic acid, allyloctadecyl ester	Dodecane	Undecane
3	Benzene,1,2,4-trimethyl	Hexadecane	Tetradecane
4	Heptadecane	Pentadecane,2,6,10,14-tetramethyl	Pentadecane
5	Heneicosane	Docosane	1H-Indene, octahydro-2,2,4,4,7,7-hexamethyl-,trans-
6	Bicyclo[4.1.0]heptane,7-butyl		Pentadecane, 2,6,10-trimethyl-
7	Dodecane,2,6,10-trimethyl		Tetratetracontane

The GC-MS data indicated the efficiency of P. aeruginosa to degrade short and medium-chain hydrocarbon. The low molecular weight of these compounds could possibly be the reason for their early elimination. A substantial decrease in the peak area of some compounds was observed which could possibly indicate a partial degradation of these hydrocarbons within the range of C18 and C24 (Figs. 1A and 1B). Likewise, the GC analysis showed the rest of the compounds remained intact after the five weeks degradation period in Table 1. This suggests the inability of P. aeruginosa to degrade long-chain hydrocarbon of more than C24 carbon.

GC-MS analysis of crude oil degradation by Pseudomonas lurida

The chromatograms and bacterial growth curve (Fig. 2A, B and C) were obtained from microcosm with a single strain of P. lurida incubated at 30°C for five weeks. The T0 chromatogram represents the crude oil profile on day one before being incubated while T5 showed the biodegradation profile of the components of crude in the fourth week when the experiment was terminated.

The GC analysis for the biodegradation of crude oil by P. lurida has indicated the elimination of many peaks of mostly less than ten carbon atoms. However, a peak identified C₂₄ i.e. tetracosane was observed to have been disappeared after the degradation period of five weeks. New peaks were also noted to have emerged at the end of the biodegradation period. These peaks indicate the presence of compounds such as Hexadecane, Hexadecane,2,6,10-tetramethyl, heneicosane, and pentacosane in Fig. 2B. The abundance of some peaks were observed to have been reduced by nearly 50% or higher when compared to the total abundance of T0 GC chromatogram and T4 GC chromatogram. This could possibly suggest a partial degradation in which the initially identified crude oil components might have been metabolized and resulted in the yield of other compounds of lower molecular weight (Figs. 2A and B as well as Table 2). P. lurida may have not eliminated most of the hydrocarbon compounds of more than C₁₂ n-alkanes but has drastically reduced the abundance of many peaks at the completion of the study. This is in addition to a compound with high molecular weight which was observed to have been eliminated at the end of the study of the microcosm. This is the first study to have reported biodegradation of crude oil by P. lurida.

Table 2

List of degraded, partially degraded and non-degraded compounds
S/No	Degraded Compounds	Partially Degraded Compounds	Not Degraded Compounds
1	Toluene	Dodecane	Undecane
2	Cyclohexane,2-propenyl-	Octane,2,6-dimethyl-	Octane,2,6-dimethyl-,
3	p-xylene	Sulphurous acid, decylpentyl ester	Tetradecane
4	Ethylbenzene	Hexacosane	Decahydro-4,4,8,9,10-pentamethylnaphthalene
5	Benzene,1,2,3-trimethyl	1H-Indene,octahydro-2,2,4,4,7,7-hexamethyl-,trans-	Pentadecane
6	Benzene,1,3,5-trimethyl-		Pentadecane,2,6,10,14-tetramethyl
7	Tetracosane		Heptadecane
8			Octadecane
9			Eicosane
10			Docosane
11			Tetratetracontane

The bacterium was also observed to have recorded the highest number of un-degraded crude oil components. Therefore, the lower growth rate of the bacterium could suggest the reason for the least elimination of crude oil components in this study. The list of the degraded hydrocarbons, partially degraded and un-degraded hydrocarbons compounds were presented in Table 2.

Results of qPCR analysis

The results of the qPCR analysis indicate the highest relative gene fold for 4-hydroxybenzoate 3-monooxygenase gene from the microcosms with a novel P. lurida (Fig. 5), followed by alkane monooxygenase gene (Fig. 6) from the study with P. aeruginosa, and lastly catechol-2,3-dioxygenase gene in a study with P. lurida (Fig. 7). The relative fold expression of genes coding for the crude oil-degrading enzymes detected by the analysis of the qPCR has indicated a colossal variation among the analysed functional genes between the two studied bacterial species. For instance, the analysis by the qPCR has successfully detected the gene expression for catechol,2,3-dioxygenase (cat23), and benzoate monooxygenase (ben) genes from the crude oil biodegradation study with P. lurida. However, these two functional genes were not detected in the similar study with P. aeruginosa. But there was a successful detection of the alkane monooxygenase (alkB) gene from the microcosms with P. aeruginosa. Similarly, a substantially higher relative fold expression of the 4-hydroxybenzoate monooxygenase gene has been recorded as 2.1x10¹⁴ fold after the first week (T1) compared to day zero (T0) of the crude oil degradation process by the novel P. lurida (Fig. 5).

Conversely, an extremely lower relative gene fold of 60.91 for the cat23 gene was observed in a microcosm with P. lurida (Fig. 6). Thus, that is the lowest gene expression fold observed from this study. In the same vein, the relative gene fold of 2156.87 was detected for the alkB gene from the microcosms with P. aeruginosa (Fig. 6). Thus, significantly higher than the detected gene expression fold for the cat23 gene and greatly lower than the Ben gene. The extremely higher relative abundance of the 4-hydroxybenzoate monooxygenase gene detected from the microcosms with P. lurida during the first week of the experiment could indicate an immediate activation of this gene following the experiment set up.

The ability of microorganisms to adopt to crude oil supplemented environment following inoculation is a great factor that determine the rate at which the microbes degrades the crude oil components (Das and Chandran, 2011). Several studies have reported efficient crude oil degradation by different bacterial species such as Bacillus, Pseudomonas, Alcaligenes, Acinetobacter, Rhodococcus, Arthrobacter, Burkholderia, Stenotrophomonas, Methylobacterium, Corynebacterium, Nocardia, and Flavobacterium (Zang et al., 2011; Okoh, 2003; Das and Chandran 2011; Chen et al., 2017). For this study, P.aeruginosa was considered because of its reported efficiency to degrades a wide range of hydrocarbons, especially crude oil in the Niger Delta and elsewhere (Okoh, 2003; Ijah and Atai, 2003; Das and Chandran, 2011; Chen et al., 2017). Since the indigenous bacterial flora in the crude oil polluted sites possesses the ability to utilize hydrocarbons as their source of carbon and energy. Therefore, P. lurida was considered for this study. The biodegradation of crude oil happens in the order of carbon atom with alkanes being the first to be degraded and then followed by branched alkanes, the aromatic hydrocarbons, and lastly cycloalkanes (Antai and Mgbomo, 1993; Sugiura et al., 1997; Ijah and Antai, 2003; Parach et al., 2017). Similarly, mixed microbial consortia could mobilize long-chain n-alkanes of up to 44 carbon (Ijah and Antai, 2003). The GC chromatograms (Figs. 1 and 2) obtained from this study have corroborated this. Interestingly, lower chain hydrocarbon was observed to have been degraded (Tables 1 and 2) in all the microcosms with individual strain and mixed consortia. The GC analysis in this study has equally indicated a drastic reduction in the area of the peak of some of the medium-chain hydrocarbons, and significant changes in the composition of most of these peaks were also observed. This could suggest a partial degradation of these compounds where some of them were metabolised to yield a lower chain of varying hydrocarbons. This observation was noted across all the soil microcosms.

The study of the literature has indicated the vulnerability of n-alkanes to microbes (Lin et al. 2014; Pan and Ma, 2019). Among these species, the exceptional utilisation of n-alkanes by the Pseudomonas strains as the sole source of carbon has been well documented (Zhang et al. 2011; Wang et al. 2017; Pan and Ma, 2019). Thus, high enzymatic ability permits the degradation of constituents of crude oil by the microbial consortia, and key knowledge about the role of microbial diversity as well as the factors aiding the role of microbial functions is necessary to effectively study biodegradation (Chioma et al., 2020). To underpin the environmental roles of these bacterial species used in the present study, three key functional genes namely; alkane monooxygenase, catechol-2,3-dioxygenase, and benzoate monooxygenase genes were studied in a soil microcosm with crude oil. For this study, these catabolic genes such as monooxygenase and dioxygenase coding for an enzyme responsible for the mineralization of the constituents of crude oil were quantified by the analysis of qPCR. The abundance of functional genes detected in correlation with oil pollution in a standard microcosm with different bacterial species could suggest the ability of a bacterial species for the bioremediation of crude oil-polluted soils. The analysis by the qPCR in this study has indicated a hugely varied fold expression across the studied microcosms.

The present study has successfully quantified the relative fold expression of the benzoate monooxygenase gene from microcosms with P. lurida and was observed to have been the highest expression fold of 2.122x10¹⁴. This same microcosm was noted to have reduced a substantial hydrocarbon among the studied microcosms. The detection of the fold expression of this gene was done during the first week of the incubation. The detected gene fold expression could suggest a rapid adaptation of P. lurida in the crude oil impacted microcosms. Similarly, the results of the GC-MS have indicated the elimination of the short and medium-chain hydrocarbons from this microcosm with P. lurida. In the same vein, the overall abundance of long-chain hydrocarbons was mostly reduced with many of them being metabolised to yield short and medium-chain hydrocarbons. However, the catechol-2,3-dioxygenase gene was detected with a very low fold expression of 60.912 from this microcosm. The detection of the cat23 gene from this microcosm with P. lurida was also done during the first week of the incubation. The catechol-2,3-dioxygenase gene produces an enzyme critical for the aromatic ring cleavage which is responsible for the biodegradation of a wide range of aromatic hydrocarbons by a range of bacterial species (Lima-Morales et al. 2016; Ehis-Eriakha et al. 2020).

The present study has observed the fold expression of this Cat23 gene from microcosms with P. lurida. The detected expression fold by the qPCR was the lowest among the studied functional gene in this study. However, the detection of the gene could suggest the environmental application of the novel P. lurida for the eco-restoration and bioremediation of crude oil-polluted soils. The detection of enzymes catechol dioxygenase during the bioremediation of oil-polluted environments has been well documented, and this could justify the critical role of Cat23 in the degradation of a wide range of hydrocarbons compounds (Thomas et al., 2016). Previous research has indicated that the Cat12 pathway triumph in low pollution conditions, whereas the cat23 pathway preferred tenacious higher conditions (Sei et al., 2004). Moharikar et al., (2003), observed that, unlike the Cat12 gene, the Cat23 gene was normally detected from the communities polluted with crude oil. Therefore, the detection of cat23 by the present study could suggest a little high load of crude oil pollution in the microcosms. Similarly, the unsuccessful quantification of cat12 in the microcosms using P. aeruginosa suggests the utilisation of metabolic routes other than ortho and meta-cleavage routes. The detection of the 4-hydroxybenzoate 3-monooxygenase gene by the present study has indicated the utilization of the protocatechuate catabolic route by P. aeruginosa for the degradation of some of the constituents of crude oil (Romero-Silva et al. 2012). Some bacterial and fungal species have been commonly known for their ability to utilize this catabolic route for the degradation of compounds such as aromatic hydrocarbons. The protocatechuate is a crucial central intermediate for the degradation of a wide range of aromatic compounds including 4HBA and 3HBA among others involving numerous bacterial species (Jimenez et al. 2002; Perez-Pantoja et al. 2008; Perez-Pantoja 2012).

The alkB gene encodes an enzyme's terminal monooxygenase during the biodegradation of crude oil (Liu et al. 2015). The primary target of this enzymes focus on degradation of short and medium-chain (C6 ~ C16) alkanes (Wang et al. 2010; Cappetti et al. 2010; Ulrich et al. 2008). Also, the alkB gene has been reported with a greater specificity for the mineralisation of varied components of petroleum hydrocarbons (Liu et al. 2015). Additionally, a previous study has indicated the enzymatic ability of the alkB gene to degrade long-chain alkanes of up to C₄₀ (Liu et al. 2015; Wasmund et al., 2009; Throne-Holst et al., 2007). This suggests that the extensive elimination of short and medium-chain hydrocarbon and the lowering of the abundance of long-chain hydrocarbons by P. aeruginosa was catalysed by the enzymes coded by the alkB gene. The biodegradation of aromatic hydrocarbons and long-chain alkanes in crude oil has been slightly achieved due to the higher hydrophobicity of these compounds (Pan and Ma, 2019). Therefore, the finding of the present study is consistent with studies reported by Liu et al. 2015 and Pan and Ma et al. 2019 which indicated the elimination of short and medium-chain hydrocarbons. Their findings also showed the utilization of more than C₁₅ hydrocarbons by the strains of P. aeruginosa which has also been noted in the present study.

This study has indicated the efficiency of the GC-MS technique and qPCR analysis as an effective approach for monitoring crude oil biodegradation. The present study has provided an insight into metabolic dynamics of both P. lurida and P. aeruginosa during the biodegradation of crude. From the results of the GC-MS, it can be concluded that these bacterial species possess the potential ability for the bioremediation of crude oil-polluted environments. It is further concluded that the present study has shown that P. lurida been the first time to have been studied for biodegradation of crude oil, could substantially mobilize a wide range of hydrocarbons on the terrestrial environment. The overall finding made by the present study has established the broad application of this novel bacterial species for the management of crude oil contaminants in the environment, which has not been reported previously. The data reported from this study could be utilized as a tool to devise an engineered crude oil bioremediation process to address the long devastating crude oil pollution in oil impacted communities across the world.

Acknowledgement

I would like to sincerely thank the Petroleum Technology Development Fund (PTDF) Nigeria for funding the PhD research programme.

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“This manuscript contained was purely based on environment studies and none of the reported information require ethical approval”

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“This manuscript did not contain any data or information that requires consent before publishing it.”

Authors Contributions

“For this study, literature survey, experimentation, data analysis and manuscript writing were conducted by Dr Mustapha Gani while Dr Mohammed Azizur Rahman supervised experiment and review the manuscript. All authors read and approved the final manuscript.”

Competing of Interest

“The authors have no relevant financial or non-financial interest to disclose.”

Funding

“Not applicable”

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No competing interests reported.

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Selection and quantification of key functional genes of some selected bacterial species in a microcosms study for biodegradation of crude oil

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Abstract

Figures

Introduction

Methodology

Sample collection and processing for the biodegradation of crude oil

Preparation of the experimental setup

Microcosms set up

Sample Preparation and Gas Chromatography-Mass Spectrometry Analysis

Real-time PCR experiment

Analysis of qPCR data

Results

Results of qPCR analysis

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1