Metataxonomic Characterization of Enriched Consortia Derived from Oil Spill-Contaminated Sites in Guimaras, Philippines, Reveals Major Role of Klebsiella sp. in Hydrocarbon Degradation

Oil spills are major anthropogenic disasters that cause serious harm to marine environments. In the Philippines, traditional methods of rehabilitating oil-polluted areas were proven to be less efficient and cause further damage to the environment. Microbial degradation has poised itself to be a promising alternative to those traditional methods in remediating oil spills. Hence, the present study aimed to enrich and characterize hydrocarbon-degrading microbial consortia from oil-contaminated regions in Guimaras Island for potential use in bioremediation. A total of 75 soil samples were obtained and used as inoculum for the enrichment for hydrocarbon degraders. Afterwards, 32 consortia were recovered and subjected to the 2,6-DCPIP assay for biodegradation ability on four types of hydrocarbons: diesel, xylene, hexane, and hexadecane. The consortia that obtained the highest percent degradation for each of the four hydrocarbons were “B2” (92.34% diesel degraded), “A5” (85.55% hexadecane degraded), “B1” (74.33% hexane degraded), and “B7” (63.38% xylene degraded). Illumina MiSeq 16S rRNA gene amplicon sequencing revealed that the dominant phyla in all consortia are Pseudomonadota (previously Proteobacteria), followed by Bacillota (previously Firmicutes). Overall, the amplicon sequence variants (ASVs) retrieved were mainly from the Gammaproteobacteria class, in which many hydrocarbon-degrading bacteria are found. Predictive functional profiling of the consortium showed the presence of genes involved in the degradation of recalcitrant hydrocarbon pollutants. Fatty acid metabolism, which includes alkB (alkane-1-monooxygenase) and genes for beta oxidation, was inferred to be the most abundant amongst all hydrocarbon degradation pathways. Klebsiella sp. is the predominant ASV in all the sequenced consortia as well as the major contributor of hydrocarbon degradation genes. The findings of the study can serve as groundwork for the development of hydrocarbon-degrading bacterial consortia for the bioremediation of oil spill-affected areas in the Philippines. Likewise, this paper provides a basis for further investigation into the role of Klebsiella sp. in the bioremediation of hydrocarbon pollutants.


Background
Oil spills are one of the main culprits in the destruction of marine ecosystems, and their occurrence is relatively understudied in the Philippines.Despite this, the country is especially vulnerable to oil leaks because of the considerable maritime trafc it receives as an archipelago.Notably, the Iloilo Strait, which separates the islands of Guimaras and Panay, serves as a common route utilized by oil tankers transporting fuel between major population centers.On 11 August, 2006, the Guimaras oil spill accident occurred that was responsible for discharging roughly 350,000 tons of fuel oil approximately 200 km from the coastal areas of Guimaras Island and the neighboring Panay and Negros islands [1].It contaminated about 450 hectares of protected mangrove forests, threatened Taklong Island National Marine Reserve [1,2], and contaminated seafood products [3,4], dubbing the incident as the most tragic oil spill in the Philippines.
In the aftermath of the Guimaras oil spill, traditional strategies such as spraying chemical dispersants and using spill booms were employed to prevent the oil from spreading further.In addition, siphoning of the oil was also done seven months after the oil spill.However, due to the vast area afected and rough sea currents, the oil decontaminating efort was unsuccessful [5].Because of the inadequate efciency and heightened costs of these strategies, bioremediation methods to remove oil spills should be explored.Bioremediation strategies for the removal of oil pollutants have proven to be efcient, cost-efective, and noninvasive compared to the physicochemical methods of detoxifying oil contaminants [6].
Autochthonous bioaugmentation utilizes native microorganisms in the polluted area for the removal of oil.Tis method for bioremediation is highly desirable as it minimizes the perturbation to the microbial ecology of the affected sites [7,8].Moreover, native microorganisms can better promote bioremediation efciency as they tend to have a higher probability of surviving in the natural environment.Overall, microorganisms can utilize oil pollutants as a source of carbon and energy, which eventually leads to the mineralization of these contaminants into water, carbon dioxide, mineral salts, and biomass.
Oil spills result in the "bloom" of hydrocarbondegrading species, which play a crucial role in the process of bioremediation.For instance, the genus Oleispira, an alkane degrader, was abundant at the site of the Deepwater Horizon blowout in the Gulf of Mexico [9].Furthermore, alkane-degrading species from Rhodobacteriaceae, Sphingomonadaceae, Chromatiales, and Actinobacteria, and polycyclic aromatic hydrocarbon (PAH) degraders, such as Pseudomonas stutzeri, Sphingomonas sp., and Tistrella mobilis, were found to be abundant along the Spanish coastline afected by the oil spill from the Prestige oil tanker [10].Due to the complexity of crude oil, bioremediation is best carried out by a community of microorganisms, with each member species responsible for the degradation of specifc types of hydrocarbon depending on their respective enzymatic machinery [11].Species involved in the bioremediation of oil from a polluted area can be enriched and then identifed with the use of metataxonomics [12], which utilizes genetic markers, such as the 16S rRNA gene for bacteria and archaea, cross-referenced to a database.In addition, predictive analysis of the functional profle based on the amplifed 16S rRNA sequences from the microbial community can aid in elucidating the role of the members of the consortium in hydrocarbon degradation.
Given that the Philippines is an archipelago, it is a hotspot for oil spill incidents that pose a substantial threat to its ecosystems.However, there is a paucity of information regarding the microorganisms suitable for bioremediation of oil spill-polluted sites in the country.Hence, the objective of this study was to enrich and characterize indigenous hydrocarbon-degrading consortia from oil spillcontaminated areas in Guimaras, an island province that is greatly impacted by oil spill incidents, for potential use in bioremediation.Soil sampling was done at 12 : 00 pm PST (Philippine Standard Time), while it was still low tide.A total of 75 soil samples were gathered, 42 in A and 33 in B. Soil samples were collected using a small shovel to a depth of 10-30 cm.Ten grams of each soil sample were taken from each sampling point.Te soil samples were placed in sterile Ziploc bags which were then put inside an ice chest.

Enrichment for Hydrocarbon-Degrading Consortia.
Te protocol for the enrichment of microbial consortia was carried out in Bushnell-Haas (BH) medium as previously described [13] with slight modifcations.Soil samples were placed in a sterile 50 ml Falcon tube containing 30 ml of BH (composition in g/L: 0.2 g MgSO 4 ; 0.02 g CaCl 2 ; 1 g KH 2 PO 4 ; 1 g K 2 HPO 4 ; 1 g (NH 4 ) 2 SO 4 ; and 0.05 g FeCl 3 ) medium.Te BH was supplemented with 1.5% (v/v) diesel as the sole carbon source.Te tubes were incubated at 30 °C with shaking at 180 rpm for 7 days.After incubation, a 1% aliquot was transferred to the new BH medium with 1.5% (v/v) diesel and was incubated under the same conditions.Te initial enrichment step was repeated for a total of three cycles (one cycle is equivalent to 7 days).After the initial enrichment, viable consortia were harvested through centrifugation (4,500 ×g, 15 min).Te pellets were resuspended in fresh BH medium supplemented with 1.5% (v/v) diesel as the sole carbon source.Tese consortia were cryopreserved inside the −80 °C biological freezer using 50% glycerol as the cryoprotectant.

Te 2,6-Dichlorophenolindophenol (DCPIP) Assay.
Te 2,6-dichlorophenolindophenol (DCPIP) assay, as previously described in [14], was performed with minimal modifcations to evaluate the ability of the enriched consortia in the degradation of hydrocarbons.Te microbial consortia were precultured in 50 ml BH medium with 1.5% (v/v) diesel and were incubated at 30 °C with shaking at 2 International Journal of Microbiology 180 rpm until the optical density at 600 nm reached 1.0.Glucose as a carbon source was the positive control.Te cultures were then centrifuged at 4000 ×g rpm for 5 min.Te pellets were then resuspended in fresh BH medium, and the optical density (at 600 nm) was adjusted to 1.0.Afterwards, the following components were added in a microcentrifuge tube: 800 μl of BH medium, 80 μl of cell suspension, 4.5 μl of 2,6-DCPIP solution (0.5% w/v), and 26.5 μl of each hydrocarbon.Te hydrocarbons tested were diesel, hexane, hexadecane, and xylene.Te negative control set-ups were prepared without inoculum.Te microcentrifuge tubes were incubated at 30 °C for 7 days.Te 2,6-DCPIP is an artifcial terminal electron acceptor that is blue in its oxidized state and colorless when it is reduced.A positive result for hydrocarbon degradation is indicated by a color change from blue to colorless, while a negative result would have no color change.To determine the percent degradation, each sample was centrifuged at 8,000 ×g for 15 minutes.Te supernatant was analyzed at 609 nm using the UV-VIS spectrophotometer.Te percentage of degradation values was computed using the formula [14] as follows: Absorbance of treated sample Absorbance of control  x 100. ( Te computed percent degradations for diesel, hexane, hexadecane, and xylene were statistically analyzed using R [15].Te following model was used for the statistical analysis: Te Shapiro-Wilk normality test was performed to assess the distribution of the data.A one-way analysis of variance (ANOVA) was conducted to assess for signifcant diferences between the percent degradations of the consortia recovered from the two sites.Post hoc analysis via Tukey multiple comparisons of means was then accomplished.(MO Bio Laboratories, Inc., California, USA).Te quantity and condition of the extracted gDNA were assessed using Victor 3 fuorometry and agarose gel electrophoresis, respectively.

Amplifcation and Sequencing of V3-V4 Region of 16S
rRNA Gene.Amplifcation of the V3-V4 region of the 16S rRNA gene and library preparation was also performed by Macrogen, Inc. (Seoul, Korea).Te V3-V4 regions of the 16S rRNA gene of prokaryotes were amplifed using primers 341F (5′CCTACGGGNGGCWGCAG-3′) and 805R (5′GACTACHVGGGTATCTAATCC-3′).Library preparation was carried out using Herculase II pipeline was used for data preprocessing and taxonomic classifcation of the sequence reads from the samples.Te sequence preprocessing step was done with the use of DADA2 denoise-paired pipeline to dereplicate sequences, remove chimeras, and to trim low-quality sequences.Chimera removal was done using the "consensus" setting of the denoise-paired pipeline.Subsequently, taxonomic assignment of representative sequences was done with the use of the SILVA 138−99 rRNA database using a Vsearch feature classifer (v2.7.0) using the default settings [16].Uncategorized reads were then subsequently fltered from the sequences.Microbial diversity metrics such as alpha and beta diversity were then subsequently generated using QIIME2 software [17,18].Te fltered outputs from DADA2 and the Vsearch classifer were exported to R using phyloseq, and compositional plots were generated using the Microbiome package [19,20].

Predictive Functional Profling of the Hydrocarbon-
Degrading Consortia via PICRUSt2.PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) (version v2.0.3-b) was used for the prediction of the functional composition of the metagenomes [21].Te method used for the predictive functional profling in environmental samples was adapted from the methodology that was previously described [22,23].Te absolute abundance table of amplicon sequence variants (ASVs) generated from DADA2 was used as the input.Taxonomic classifcations of each ASV were derived from the output from the Vsearch classifer.Te "-stratifed" option was added to the PICRUSt2 full pipeline to generate the individual contribution of each ASV for every predicted metabolic pathway.Postprocessing and visualization were carried out using R Studio [20].Processing was performed using dplyr and qiime2r, while visualization was accomplished with the use of ggplot2, heatmap.2, and microbiome [21,22].

Hydrocarbon Degradation by the Enriched Microbial
Consortia.Tirty-two hydrocarbon-degrading consortia (16 each from sites A and B) were recovered from oil spillafected sites after three cycles of enrichment cultivation on BH medium supplemented with 1.5% diesel, suggesting their ability to utilize diesel as a sole carbon source.Te absence of growth on the other 43 consortia could be attributed to the limitations during the enrichment step (i.e., concentration of diesel, incubation conditions, salinity, and pH of the culture medium) or the inability to grow on the hydrocarbon.Te 2,6-DCPIP assay revealed that all enriched consortia exhibited degradation activity on the four hydrocarbons tested.Te consortium deemed as the best degrader for each of the hydrocarbons, and the respective percent degradation values were as follows: Consortium B2 for diesel (92% degraded), A5 for hexadecane (86% degraded), B1 for hexane (74% degraded), and B7 for xylene (63% degraded) (Figure 2).One-way ANOVA showed that between the two sites, only the percent degradations of the consortia during the diesel treatment had no signifcant diferences (P � 0.745 > 0.5), while the rest of the hydrocarbon treatments produced a p value that is less than 0.5.Furthermore, post hoc analysis via Tukey's honest signifcant diference for hexane, hexadecane, and xylene revealed that consortia derived from site B had a higher overall percent degradation as compared to site A.

Diversity Metrics of A5, B1, B2, and B7.
Consortium B7 contained the most varied species composition amongst all tested consortia, followed by B2 and B1 (Table 1).Consortium A5 had the least diverse species composition.Rarefaction curves revealed that all consortia were sufciently sequenced and have captured their entire diversity.Tis was also supported by Good's coverage (C) values.Tis proves that all species detected in the four consortia make up 100% of the population.Te Bray-Curtis dissimilarity index (Table 2 and Figure 4) suggests a high degree of similarity in the metataxonomic profles amongst the consortia.

Predictive Functional Profles of A5, B1, B2, and B7.
Predictive functional profling revealed that all enriched consortia were equipped with genetic machinery to degrade xenobiotic compounds, including petroleum hydrocarbons.Te mean weighted Nearest Sequenced Taxon Index (NSTI) of each of the 16S rRNA gene samples was 0.038 ± 0.007, indicating a high concordance between the reference sequences used by PICRUSt2 and the amplifed 16S rRNA gene samples.A total of 190 KEGG orthologs (KOs) related to hydrocarbon degradation pathways were predicted to be present, namely, ko00627 aminobenzoate (30 KOs), ko00361 chlorocyclohexane and chlorobenzene (7 KOs), ko00362 Notably, both pathways for aliphatic and aromatic hydrocarbons were predicted by PICRUSt2 in A5, B1, B2, and B7.Fatty acid metabolism, which includes alkB (alkane-1monooxygenase) and genes for the beta oxidation pathway, was found to be the most dominant amongst all hydrocarbon degradation pathways.Other degradation pathways inferred were ascribed to benzoate, aminobenzoate, chlorocyclohexane, chlorobenzene, chloroalkene, chloroalkane, nitrotoluene, styrene, and caprolactam.PAH hydrocarbon degradation (% RPA � 0.008) was only present on B7, the best degrader for xylene.Furthermore, it was inferred that the phylum Pseudomonadota, specifcally Klebsiella sp., mainly contributed to the potential ability to degrade different types of hydrocarbons (Figure 6).

Discussion
Microorganisms are key players in the biodegradation of hydrocarbon contaminants.Te present study enriched hydrocarbon-degrading bacterial consortia from oilpolluted regions in Guimaras Island using BH medium supplemented with 1.5% diesel as the sole carbon source.Te study demonstrated that all enriched consortia exhibit degradative activity to diesel, hexane, hexadecane, and xylene.Te highest range of percent degradation (40% to 92%) was obtained when diesel was used as the sole carbon source, which was expected as the consortia were preconditioned in   International Journal of Microbiology a diesel-containing medium during the enrichment process.Hexane, a short-chain n-alkane, was also degraded by the consortia with percent degradations ranging from 45% to 74%.Meanwhile, a larger overall percent degradation (61% to 87%) than hexane was obtained on hexadecane, a longer aliphatic hydrocarbon.Previous studies have argued that petroleum hydrocarbons with 5-10 carbon atoms are more detrimental to microorganisms since these compounds can destroy the organisms' lipid membranes [24].Xylene, an aromatic hydrocarbon, was the most recalcitrant to biological degradation amongst the four tested hydrocarbons, with percent degradations ranging from 22% to 63% only.In general, aromatic hydrocarbons resist the biodegradation process and typically involve a multistep pathway that involves the addition of reactive side chains and the subsequent de-aromatization and ring cleavage [25].16S rRNA gene sequencing revealed that Pseudomonadota species are well represented in the consortia, followed by Bacillota.Members of the phylum Pseudomonadota were proven to be pioneer oil degraders.A number of studies have revealed that Pseudomonadota is the most abundant phylum in environments contaminated with petroleum and in wastewater samples from petrochemical industries [26,27].Overall, ASVs retrieved were ascribed International Journal of Microbiology mainly to the Gammaproteobacteria class, whose dominance in enriched consortia is attributed to the fact that many hydrocarbonoclastic bacteria responsible for the frst steps of hydrocarbon degradation are found in this class [28].Species of the phylum Bacillota were highly ubiquitous and are considered as late stage-pioneer degraders of the more recalcitrant forms of hydrocarbons [29].
Te fndings of this study suggest a key role of Klebsiella sp. in hydrocarbon degradation based on its dominance in all the consortia.Consistent to this, Klebsiella sp. was previously reported to produce dioxygenases and hydroxylases that can break down alkanes [30].Moreover, it can degrade a wide range of aliphatic and aromatic hydrocarbons and can produce biosurfactants [31].Another study reported that oil feld-adapted K. pneumoniae exhibited >90%, >70%, and >40% biodegradation rates for C10-C20, C21-C22, and C31-C32 hydrocarbons, respectively [32].Notably, Klebsiella are known nitrogen-fxers, and studies have demonstrated that nitrogen-fxing hydrocarbon-degrading bacteria are significant members of consortia being used for oil bioremediation, since nitrogen is a common limiting compound in oil-contaminated sites [33].
Aside from Klebsiella sp., other ASVs of note include P. aeruginosa (A5, B7, and B2), Acinetobacter sp.(B1, and B7), Serratia sp.(B2 and B7), and C. aquatica (B1), which were previously reported in other studies as biosurfactant producers.Production of biosurfactants is advantageous in enhancing the biodegradation process as they increase the bioavailability of oil [34].P. aeruginosa cells can use petroleum-based hydrocarbons as a carbon source.A. baumannii can degrade total petroleum hydrocarbon fractions of crude oil and is found to be a more vigorous biodegrader of crude oil than other biosurfactant-producing bacteria [35].Similarly, another study [36] demonstrated that A. calcoaceticus can degrade aliphatic alkane hydrocarbons.Likewise, aerobic pathways for the removal of BTEX (benzene, toluene, ethylbenzene, and xylene) compounds are present in Serratia sp.[37].C. aquatica was isolated previously from oil-polluted sites of Mumbai Harbor [38].In addition, Achromobacter sp.(B2 and B7) and Stenotrophomonas sp.(B1, B2, and B7) are prolifc PAH degraders.Te former is a known degrader of polyaromatic hydrocarbons, particularly phenanthrene, naphthalene [39,40], and chrysene [41], while the latter can degrade high molecular weight PAHs including phenanthrene [42], pyrene, benzopyrene, and benzofouranthene [43].16S rRNA sequencing also identifed strictly anaerobic species.Te incubation conditions of BH broth, which became static at some point, must have infuenced the presence of anaerobes.For instance, Anaerospora sp.(B2 and B7) is known to degrade chlorinated hydrocarbons, particularly aryl halides like chlorobenzene and chlorophenol.In one study, it was found to be the dominant member in the consortium isolated from sediments near an oil refnery at Yanbu Industrial City, KSA [44].However, the presence of these abovementioned ASVs in the enriched consortia and their predicted degradation pathways contribution are seen to play a minor role only as compared to Klebsiella sp., whose relative abundances dominate across all sequenced consortia.Species of bacteria possess distinct activities towards diferent types of hydrocarbon contaminants, which make a consortium more advantageous in the bioremediation eforts of petroleum hydrocarbons as opposed to monocultures.Consortia have a variety of catabolic genes, and the synergistic efects of these genes are favorable to attaining purifcation of hydrocarbon pollutants [33].Furthermore, diversity metrics of each consortium as well as predictive functional profling suggest that "generalist" microorganisms predominate across all sequenced samples.Generalist hydrocarbon degraders are species that can utilize other carbon sources to support their growth as opposed to obligate hydrocarbon-degrading bacteria (OHCBs) such as Alkanivorax sp. and Cycloclasticus sp.Because of the wider range of carbon sources that they can utilize, generalists tend to outcompete the OHCBs [45].
Te number of ASVs detected in all four consortia in this paper was smaller compared to other enriched oil-degrading communities in other studies [13,23], probably due to the higher fnal concentration of diesel used during enrichment.Enrichment processes result in a signifcant decline in the diversity index of the community, mainly due to a strong selection for hydrocarbon-degrading species.Te species in the community are modifed to favor the growth of those members that can survive in the modifed environmental condition.In the same light, the toxic impacts of petroleum hydrocarbons could further reduce species richness, evenness, and phylogenetic diversity [46].Tis is primarily linked to the disruption of the biogeochemical cycle of nitrogen because functional genes and species involved in nitrifcation are signifcantly lowered [23].In addition, the limited oxygen concentration during incubation may have provided a selective advantage to certain microorganisms within the consortia.Tus, high-throughput screening methods may be utilized to enrich the functional resources of hydrocarbondegrading bacteria.Meanwhile, the low dissimilarity indices amongst the taxonomic profles of the consortia could be attributed to the similarity of the environments in which the consortia were derived from, as well as the uniform enrichment treatments that were carried out for all obtained samples.
Diverse genes for catabolism of aliphatic and aromatic hydrocarbons, especially diferent monooxygenases and dioxygenases, were predicted by PICRUSt2 in the consortia.Tese results corroborate the fndings of another study [23] which by using PICRUSt2, predicted genes responsible for the degradation of aromatic hydrocarbons, including chlorobenzene, naphthalene, PAHs, ethylbenzene, benzoate, and xylene, in their enriched microbial consortia from similarly contaminated areas.By comparing the functional profles of the natural to enriched microbial communities, the authors also found a signifcant increase in the hydrocarbon degradation pathways of enriched communities.Tis proves the importance of the enrichment process in generating a robust consortium equipped with the desired metabolic machinery.Moreover, complete degradation of hydrocarbon contaminants requires the collective eforts of diferent species in a microbial community.Overall, the International Journal of Microbiology PICRUSt2 pipeline suggests that bacterial communities in oil spill-afected areas adjust their metabolic pathways to hydrocarbon degradation.
Te paucity of research on the prokaryotic communities and their functional diversity in oil spill-polluted areas in Guimaras makes it untenable to deduce the complete picture of the consortium dynamics from the time of the oil spill incident up to the present day.Assessing the microbial community profles and their corresponding functional diversity is important in evaluating the recovery of oil spill-afected areas.For instance, the recuperation of the contaminated areas due to the Hebei Spirit oil spill incident in South Korea in December 2007 was determined by examining the community successions together with their respective functional profles in the years 2014 and 2016.Te fndings for the aforementioned years demonstrated a higher abundance of microbial taxa associated with total petroleum hydrocarbon (TPH) degradation in 2014, but the taxa associated with PAH degradation were similar both years [47].

Conclusion
Bioremediation is a promising strategy to rehabilitate oil spill-afected areas.In this method, the metabolic potential of microorganisms is harnessed to degrade these toxic pollutants.Te present study investigated consortia enriched from soil samples recovered from oil spill-afected sites in Guimaras, Philippines.Te recovered consortia exhibit signifcant activity of degrading diferent classes of hydrocarbons, namely, diesel, hexane, hexadecane, and xylene, after multiple rounds of enrichment.Moreover, 16S rRNA gene sequencing showed that these consortia were composed primarily of generalist hydrocarbon degraders, with Klebsiella sp.dominating all characterized consortia.Predictive functional profling using PICRUSt2 also inferred the presence of a number of relevant catabolic pathways for aliphatic and aromatic hydrocarbon degradation.Klebsiella sp.contributed mainly to the hydrocarbon-degrading capacity of each characterized consortium.Tis study, in turn, highlights the development of indigenous microbial consortia for the bioremediation of oil spill-afected areas.Likewise, this also warrants further investigation as to the specifc functions of the member species in the consortia, especially Klebsiella sp., in the degradation of petroleum hydrocarbons.Lastly, additional research should incorporate de novo metagenomic approaches to study the potential functional profle of the enriched consortia from Guimaras, Philippines, with emphasis on their role in the degradation of petroleum hydrocarbons.
Hydrocarbon-Degrading Consortia 2.4.1.Genomic DNA Extraction from the Hydrocarbon-Degrading Consortia.Total genomic DNA (gDNA) extraction from the consortium which obtained the highest percent degradation for each hydrocarbon tested was performed by Macrogen, Inc. (Seoul, Korea), following the protocol of the UltraClean ® Microbial DNA Isolation Kit

Figure 2 :Figure 3 :
Figure 2: Box plot showing the percent biodegradation of the hydrocarbon substrates tested on the enriched hydrocarbon-degrading consortium via the 2,6-dichlorophenolindophenol (DCPIP) assay.Te consortia were classifed according to their site of origin.Bottom and top error bars indicate the 25th and 75th percentile per each degradation assay, respectively.

Figure 6 :
Figure 6: Prediction of the relative contribution of each ASV, binned according to phyla (a) and genus/family (b) for the hydrocarbon degradation pathways present in A5, B1, B2, and B7.Te indicated counts shown in the plot were derived from the "Relative taxon abundance" column from the stratifed PICRUSt2 output, which is the ASV's relative abundance in a given sample multiplied by the predicted function count found in the reference genome mapped to a given ASV.(a) Function relative abundance (per phylum).(b) Function relative abundance (per genus).
Te average soil temperature and pH during the time of sampling were 30 °C ± 1 °C and 7.1 ± 0.2 in Site A, while 30 °C ± 1 °C and 7.3 ± 0.1 in Site B.