What if the best way to break down stubborn oil pollution is not to find one super microbe, but to build a compatible microbial team? This study tests that idea with fungi and bacteria from contaminated soil. Bioremediation uses microorganisms as a cost-effective alternative for restoring areas impacted by persistent contaminants such as polycyclic aromatic hydrocarbons, or PAHs.

The study explored hydrocarbon-degrading microbial consortia by prospecting native species from soils contaminated with a blend of twenty percent biodiesel and eighty percent diesel. After enrichment in minimal medium containing diesel oil as the sole carbon source, gene sequencing identified seven fungi and twelve bacteria.

The drop collapse test indicated that all fungal strains and four bacterial strains produced biosurfactants with at least twenty percent surface-tension reduction, while extracellular laccase activity was especially high in Stenotrophomonas maltophilia P05R11. After antagonistic testing, four compatible consortia were formulated, and the best mixed consortium removed ninety-three point six one percent of total PAHs and ninety-three point five two percent of TPH from C5 to C40.

Accidental spills are frequently associated with activities involving the petroleum-product supply chain, and exposure can cause irreversible impacts on organisms and ecosystems. Crude oil contains a complex mixture of hydrocarbons, asphaltenes, and resins, including polycyclic aromatic hydrocarbons.

PAHs persist because of low solubility, high molecular weight, and the stability created by fused aromatic rings. Their bioaccumulation is associated with cytotoxic, carcinogenic, mutagenic, and teratogenic effects, strengthening the need for environmental remediation and restoration.

Sustainable remediation technologies have received increasing attention in response to this environmental challenge. Microbial bioremediation has advantages over physicochemical techniques associated with complex pretreatments, inefficient removal of residual contaminants, and high overall cost.

This efficient, low-cost biological system can mediate biotransformation, detoxification, and permanent removal of organic contaminants, including high-molecular-weight PAHs. However, establishing a system that promotes contaminant consumption and mineralization requires selecting microorganisms with appropriate metabolic mechanisms.

A single microorganism typically metabolizes only a limited range of hydrocarbons. Synergism through complementary biochemical pathways allows one organism to degrade a metabolite produced by another, helping complete hydrocarbon degradation or alleviate toxic and inhibitory effects.

The primary objective was to develop microbial consortia with potential for petroleum-hydrocarbon bioremediation. Fungal and bacterial strains were isolated from experimental areas intentionally contaminated with B20 biodiesel: twenty percent biodiesel and eighty percent diesel.

After identification, consortium combinations were designed using screening results for extracellular laccases, biosurfactants, and species compatibility. The consortia and individual strains were then tested for diesel-oil degradation potential, including Trichoderma koningiopsis, evaluated in this context for the first time.

Samples came from the Ressacada Experimental Farm in Florianópolis, southern Brazil, where controlled fuel and biofuel contamination had been monitored for twenty-five years, from nineteen ninety-eight to two thousand twenty-three. Historical analytical data led to selecting areas P05 and P10 to prospect petroleum-hydrocarbon-degrading microorganisms and analyze current contaminant concentrations.

Area P05 had received one hundred liters of B20 biodiesel made with soybean biodiesel and diesel, followed by natural attenuation remediation starting in two thousand eight. Area P10 had received one hundred liters of B20 biodiesel made with palm biodiesel and diesel, followed by bioaugmentation combined with iron oxide in two thousand seventeen.

Soil samples were collected on April twenty-eighth, two thousand twenty-one, using a Dutch auger, with three perforations near each contamination source well. Samples were collected at one point five meters for area P05 and one point eight meters for area P10, based on previous data about contaminant-release depth.

The samples were homogenized into mixed soil compositions, and approximately five hundred grams kept at four degrees Celsius were sent to the laboratory. The isolation and selection of fungi and bacteria used enrichment in minimal medium, with diesel oil as the only carbon source.

Five grams of contaminated soil were transferred to flasks containing eighty milliliters of Bushnell Haas medium enriched with one percent filtered commercial diesel oil. The flasks were shaken at one hundred fifty revolutions per minute and twenty-eight degrees Celsius, with samples collected after seven and fourteen days.

The enriched cultures were serially diluted, plated on Nutrient Agar and Malt Extract Agar, and incubated for seven days at approximately twenty-eight degrees Celsius. Colonies selected by morphology, size, and coloration were restreaked for purity and subjected to a second enrichment with five percent diesel oil.

Genomic DNA from fungi and bacteria was extracted using the Plant and Fungi DNA Isolation Kit and the DNeasy Kit, respectively, and DNA concentration and purity were assessed with a NanoVue Plus spectrophotometer. Fungal identification amplified partial regions of the beta-tubulin gene using the Bt2a and Bt2b primers, and partial regions of the Calmodulin gene using the CMD5 and CMD6 primers.

These genes were selected because they provide higher resolution for distinguishing closely related fungal species, particularly within Aspergillus and Penicillium. To evaluate biosurfactant production, twelve bacterial strains were first cultivated for forty-eight hours in nutrient broth.

One percent of the inoculum was transferred to Mineral Salts Medium containing its listed salts and trace-element solution. The medium was supplemented with three percent filtered soybean oil and incubated at one hundred thirty revolutions per minute and thirty degrees Celsius for seven days.

After incubation, cultures were centrifuged at four thousand five hundred revolutions per minute for fifteen minutes, and the supernatant was used for subsequent tests. Surface tension was measured using approximately three milliliters of culture supernatant and a goniometer two hundred fifty-F-one.

The goniometer was calibrated with distilled water, and the pendant-drop curvature was analyzed with DROPimage Advanced software. Ten measurements were taken at one-second intervals for each reading, averaged, and performed in triplicate at room temperature. Surface-tension reduction was calculated from the negative variation during cultivation relative to the negative control, multiplied by one hundred.

All isolated fungal and bacterial strains first underwent qualitative laccase screening to identify candidates for quantitative analysis. Mycelial disks from seven fungal strains were used as inoculum in modified Czapek Yeast Extract broth. The medium contained one hundred fifty micromolar copper sulfate, and the flasks were shaken at one hundred twenty revolutions per minute and twenty-eight degrees Celsius for twelve days.

Based on biosurfactant and laccase assays, six bacterial strains and five fungal strains were selected for growth-dynamics analysis during co-cultivation. The purpose of this step was to formulate microbial consortia capable of degrading petroleum hydrocarbons.

Diesel-oil biodegradation, including PAHs and TPH, was evaluated in flasks containing eighty milliliters of Bushnell Haas medium and one percent diesel oil. The experiment compared mixed consortia and individual strains, including the FFB1, BB1, FF1, and FBB1 combinations.

Statistically significant differences among experimental treatments were analyzed with analysis of variance followed by the Scott–Knott multiple-comparison test. This analysis provided the statistical test used to compare the experimental treatments.

Even a decade after contaminants were released in area P05, residual concentrations of numerous PAHs and significantly elevated BTEX levels remained. The analysis used National Environment Council guidelines from Resolution number four hundred twenty of two thousand nine to establish soil-contamination concentration limits.

Benzene, toluene, and xylenes exceeded recommended soil limits in P05, while naphthalene and benzo(a)anthracene were the PAHs above established limits. In area P10, contaminated in two thousand seventeen, xylenes, toluene, naphthalene, and anthracene exceeded the recommended average.

Table 1 reports gas-chromatography measurements of hydrocarbons in soil from areas P05 and P10, alongside concentration limits established by CONAMA. It includes BTEX compounds and multiple polycyclic aromatic hydrocarbons, with several entries marked “not reported” where no limit is provided.

The table matters because the authors use these measured concentrations to document residual contamination, including contaminants detected a decade after the release in area P05. Nineteen isolates remained viable after two enrichment stages in minimal mineral medium containing commercial diesel oil as the sole carbon source.

Eight isolates originated from area P05 and eleven from area P10, using the P, R, and isolate-number nomenclature. Morphological analyses separated the isolates into seven fungi and twelve bacteria. Table two identifies the closest fungal matches for seven strains using partial sequences from the beta-tubulin, or BenA, and Calmodulin, or CaM, genes, alongside their GenBank accession numbers.

The results include six Penicillium species and one Trichoderma species, consistent with the paper’s broader classification of these isolates within Ascomycota. Notably, P05R2 matches Trichoderma koningiopsis, while the remaining strains are assigned to Penicillium taxa, including P.

janthinellum, P. pulvillorum, and P. simplicissimum. Table three identifies twelve bacterial strains by partial sixteen-S rRNA sequencing, pairing each strain with its closest NCBI match and a GenBank accession number.

The table includes members of Proteobacteria and Firmicutes, with Burkholderia and Bacillus appearing across both sampling areas, P05 and P10. It also records distinct representatives such as Dyella japonica, Paraburkholderia sp., Stenotrophomonas maltophilia, and Serratia marcescens, providing the taxonomic basis for linking isolates to later analyses of bacterial groups and biosurfactant activity.

Nineteen isolates—seven fungi and twelve bacteria—were screened for biosurfactant production in minimal salt medium using soybean oil as the sole carbon source. Surface-tension reduction was used to select strains for later stages because increased reduction indicates surfactant activity.

The control medium had an average surface tension of fifty-four point five seven millinewtons per meter. The supernatant of every fungal culture showed significant surfactant capability, with surface-tension reduction greater than twenty percent.

Figure 2 shows surface-tension reduction for nineteen fungal and bacterial isolates grown in minimal salt medium with soybean oil. All fungal culture supernatants exceeded the twenty-percent screening threshold, while the chart also identifies statistically distinct groups using letters from the Scott–Knott analysis, with a p-value below zero point zero zero zero one.

The authors used these reductions to select strains for subsequent biosurfactant studies. The preliminary laccase screen produced a greenish-blue color shift in four fungal strains and five bacterial strains. Only isolates with that positive color change were selected for quantitative analysis of extracellular laccase activity.

Among the selected fungi, the first quantitative enzymatic activity appeared after three days in P. janthinellum P05R3, at zero point one six seven units per milliliter. Figure three tracks extracellular laccase activity for four fungal strains, P05R1, P05R2, P05R3, and P10R5, across fourteen days of cultivation.

The chart shows that activity appears at different times and fluctuates over the monitoring period; P05R2 reaches a plotted peak of about zero point four three U/mL on day thirteen. This time-course matters because it identifies when fungal isolates produce the enzyme most actively, supporting the quantitative follow-up after the initial color-screening assay.

Figure four tracks extracellular laccase activity alongside bacterial growth over six days. Solid lines show laccase activity in units per milliliter, while dashed lines show optical density at six hundred nanometers, a spectrophotometric measure of growth.

The authors report that all bacterial strains reached their enzymatic peak within the first twenty-four hours, with Stenotrophomonas maltophilia P05R11 reaching zero point nine one seven units per milliliter. Growth-dynamics analysis revealed moderate antagonistic effects that partially inhibited colony development and more intense antagonistic activity that completely inhibited culture growth.

Compatible consortia were selected by choosing species that could grow together in minimal medium with one percent diesel and showed no antagonistic interactions. The resulting combinations were FFB1, BB1, FF1, and FBB1, with their fungal and bacterial members specified in the study.

Table 4 screens pairwise compatibility across bacteria–fungus, bacteria–bacteria, and fungus–fungus combinations. A minus sign indicates no inhibition, one plus indicates a moderate antagonistic effect, and two plus signs indicate strong antagonism with complete growth inhibition.

These interactions matter because the authors used them to identify strain combinations capable of growing together when formulating microbial consortia. After fifteen days in Mineral Salts Medium, degradation rates of total hydrocarbons and PAHs differed greatly among treatments.

Four-bromofluorobenzene and chrysene were exceptions, reaching ninety-nine point nine zero percent and one hundred percent degradation by all strains and consortium combinations. The individual Serratia marcescens P10R19 strain removed pyrene at ninety-two point zero eight percent, anthracene at eighty-two point six zero percent, and one-methylnaphthalene at ninety-three point eight zero percent.

Serratia marcescens P10R19 degraded seventy-seven point seven seven percent of TPH from C5 to C40 and seventy-four point four eight percent of total PAHs, compared with forty-five point nine five and thirty-six point six zero percent for Stenotrophomonas maltophilia P05R11.

Combining those two bacterial strains in BB1 increased degradation to eighty-seven point zero five percent for total PAHs and ninety-three point nine seven percent for TPH from C5 to C40. The mixed fungal–bacterial consortium FFB1 removed ninety point four one percent of total PAHs and eighty-three point seven seven percent of TPH from C5 to C40.

FFB1 achieved higher degradation of fluoranthene, at ninety-nine point zero three percent, and fluorene, at ninety-four point eight nine percent, compared with BB1 and FF1. The best-performing consortium was FBB1, containing Trichoderma koningiopsis P05R2, Serratia marcescens P10R19, and Burkholderia cepacia P05R9.

According to the heatmap, FBB1 achieved degradation rates of at least ninety-one percent for every analyzed PAH, indicating a wide spectrum of degraded compounds with significantly higher rates. Table five is a heatmap of degradation percentages for individual hydrocarbons, total PAHs, and TPH from C5 to C40 across eight sample conditions.

The authors connect these measurements to broad hydrocarbon removal, including 83% for total PAHs and TPH, while noting persistent contamination in area P05. The strongest performer was a mixed fungal–bacterial consortium, which degraded at least ninety-one percent of every analyzed PAH and removed more than ninety-three percent of total PAHs and TPH.

The result supports microbial partnerships as a promising remediation strategy.