Degradation of a mixture of 13 polycyclic aromatic hydrocarbons by commercial effective microorganisms

3.1.1 Acenaphthylene

On Day 4 of the study, the acenaphthylene concentration ranged between 0.052 and 0.089 mg/kg. On Day 14 of the study, the acenaphthylene concentration was the highest in the control samples and amounted to 0.02 mg/kg, while it reached the lowest level in the samples with Formulations 4 and 5, amounting to 0.013 mg/kg. On the last, 35th day of the study, the acenaphthylene concentration was also the highest in the control samples (0.014 mg/kg), and it dropped to the minimum level in the samples treated with Formulations 4 and 5 (0.005 mg/kg) (Figure S1).

The use of all EM formulations accelerated the acenaphthylene degradation, when compared to the control. The achieved degradation was the lowest with Formulation 7 (yeast mix – 87.5%) and the highest (92.7%) with Formulation 2, containing bacteria from Arthrobacter, Bacillus, Cellulomonas, Paenibacillus, Pseudonocardia, Streptomyces, Pseudomonas, and Rhodococcus genera (it increased the rate of the acenaphthylene decomposition by 10.1% versus the control) (Table 3).

Rocha et al. analyzed the influence of Pleurotus ostreatus (the oyster fungus, edible mushroom) on the acenaphthylene degradation in the sandy soil. P. ostreatus degraded 57.7% of this compound in the soil enriched at the level of 30 mg/kg, and 65.8% of acenaphthylene when the soil was enriched at the level of 60 mg/kg, after the incubation period of 15 days [63].

Barnes et al. investigated the degradation of crude oil and associated PAHs using ten fungal cultures isolated from the aquatic environment: Penicillium citrinum, Acremonium sclerotigenum, Aspergillus polyporicola, Aspergillus versicolor, Fusarium equiseti, Fusarium sp., Aspergillus sp., Aspergillus favus, and Aspergillus sydowii. The studies were conducted in 20 mL of the mineral salt medium (MSM) containing 1% (w/v) crude oil as the sole source of carbon for isolates. The experimental flasks were incubated at 28°C with constant shaking at 80 rpm, for 23 days. Among the ten isolates studied, 100% of acenaphthylene was removed from six isolates [64].

3.1.2 Fluorene

On Day 4, the fluorene concentration ranged from 0.031 to 0.072 mg/kg. On Day 14, the noted substance concentration was the highest in the control samples, amounting to 0.013 mg/kg, and the lowest in the samples treated with Formulations 4 and 5, of 0.008 mg/kg. On Day 35 of the experiment, fluorene was at the highest concentration, of 0.004 mg/kg, in the control samples and after application of Formulations 1 and 2, and at the lowest level, of 0.002 mg/kg, in the samples treated with Formulation 3 (Figure S10).

Fluorene proved to be a persistent substance. After the use of Formulations 5, 6, and 7, its degradation was lower versus the control (91.8–92.0%). The highest degradation was obtained for Formulation 3, at the level of 95.5% (Table 3).

Barnes et al. studied the degradation of the fluorene using ten fungal cultures, isolated from the aquatic environment: P. citrinum, A. sclerotigenum, A. polyporicola, A. versicolor, F. equiseti, Fusarium sp., Aspergillus sp., A. favus, and A. sydowii. Among the ten isolates studied, the degradation level ranged from 53.7 to 100% with Aspergillus sp., after the incubation period of 23 days [64].

Bankole et al. investigated the fluorene degradation efficiency of the marine derived filamentous fungus, Mucor irregularis, bpo1 strain. Optimization of the vital constituents of the MSM used in the study resulted in the fluorene degradation at the rate of 79.8%. The enhanced fluorene degradation efficiency (82.5%) was recorded when the optimized process variables were subjected to growth-linked validation experiments. Furthermore, the activity of enzymes, including laccase, manganese peroxidase, and lignin peroxidase, was demonstrated [65].

Nam et al. researched the influence of Sphingobacterium sp. KM-02, isolated from the soil polluted with PAHs near a mine-impacted area, on the fluorene degradation. During culturing of microorganisms with fluorene as the only source of carbon, decomposition of 78.4% of that PAH occurred within 120 h. Additionally, Sphingobacterium sp. KM-02 ability to biodegrade fluorene at a level of 100 mg/kg in the soil in the laboratory conditions was verified. During 20 days of the experiment, 65.6% of fluorene was decomposed [66].

Forty-seven fungal strains were isolated from the soil polluted with PAHs and the fluorene degradation rate was verified. The most productive of isolated strains was Absidia cylindrospora, and it was found that over 90% of fluorene was degraded within 288 h, while the process required 576 h when no microorganisms were present [67].

3.1.3 Anthracene

Another studied substance was anthracene, and on Day 4, its concentration was in the range from 0.028 to 0.072 mg/kg. On Day 14, the noted substance concentration was the highest in the control samples, amounting to 0.017 mg/kg, while it was the lowest, of 0.008 mg/kg, in the samples treated with Formulation 5. On the last day of the study, anthracene also was at the highest concentration in the control samples, of 0.005 mg/kg, and at the minimum level of 0.002 mg/kg in the samples treated with Formulations 3, 6, and 7 (Figure S2).

Also in the case of anthracene, EM formulations accelerated its degradation, and in their presence, it ranged between 91.4 and 95.1%. The anthracene degradation was the highest following the treatment with Formulation 3, which contained eight bacteria strains from the Bacillus genus (Table 3).

The influence of microorganisms isolated from the soil long polluted with creosote oil on the anthracene degradation was studied by Smułek et al. The level of this compound degradation ranged from 30 to 70%. The highest degradation was achieved for Pseudomonas mosselii and Pseudomonas mendocina [68].

Krivobok et al. isolated from the soil 39 strains of Micromycetes fungi, described as effectively degrading PAHs. Nineteen of them degraded at least 50% of anthracene. The highest degradation was obtained for Rhizopus arrhizus – 95% and Cryphonectria parasitica – 96%. Furthermore, studies were also conducted on other fungal species able to degrade anthracene, including Rhizoctonia solani (86%), Ceriporiopsis subvermispora (88%), Oxysporus sp. (94%), Cladosporium herbarum (85%), Drechslera spicifera (79%), Verticillium lecanii (77%), Coniothyrium sporulosum (57%), and Cunninghamella spp. (78–87%) [69].

Two strains of bacteria able to degrade anthracene – Ralstonia pickettii JANC1A and Thermomonas haemolytica JANC2B, were isolated and identified. They were isolated using a method for enrichment of the contaminated soil in the mineral medium. The initial anthracene concentration amounted to 100 mg/kg. It was demonstrated that 50 and 75% of anthracene was degraded after 8 and 20 days, respectively [70].

Wu et al. investigated the anthracene degradation by fungi isolated from the environment of PAH-contaminated mangrove sediments in Ma Wan, Hong Kong. Anthracene at the concentration of 50 mg/L was added to the MSM for initial screening of PAH-degrading fungi, and finally two fungal species capable of using the studied PAH as the sole source of carbon were isolated and identified as Fusarium solani – MAS2 and MBS1 strains. Anthracene removal reached 40% of the added amount after 40 days of incubation in samples with MAS2 strain [71].

3.1.4 Phenanthrene

On Day 4, the phenanthrene concentration ranged from 0.03 to 0.067 mg/kg. On Day 14, the highest substance level was 0.016 mg/kg and it was noted in the control samples, while its concentration was the lowest, of 0.009 mg/kg, in Formulations 4 and 5. On Day 35 of the study, phenanthrene reached the highest concentration of 0.008 mg/kg in the control samples, and the lowest, of 0.004 mg/kg, in the samples treated with Formulation 4 (Figure S12).

The use of Formulations 5, 6, and 7 slowed the phenanthrene degradation process versus the control level; however, these differences were not statistically significant for Day 35. The remaining formulations accelerated the degradation. The highest degradation, of 90.8%, was achieved with Formulation 1 (Table 3).

The phenanthrene biodegradation by Bacillus. licheniformis STK 01, Bacillus subtilis STK 02, and Pseudomonas aeruginosa STK 03, microorganisms that were isolated from an oil spill site, was studied. The phenanthrene concentration amounted to 50 mg/kg of the soil. After the experiment period of 60 days, 91.43, 84.83, and 83.97% of phenanthrene were decomposed by B. licheniformis, B. subtilis, and P. aeruginosa, respectively. When B. licheniformis and B. subtilis were used, 90.34% of phenanthrene was decomposed [72].

Nzila et al. isolated and characterized two bacterial strains able to degrade phenanthrene – Pseudomonas citronellolis PHC3Z1A and Stenotrophomonas maltophilia JPHC3Z2B. The initial phenanthrene concentration (C ₀ = 100 ppm) was degraded in 50% after 7 days and in 75% after 15 days [70].

A fungus, Fusarium sp., was isolated from soils polluted with crude oil (Liaohe Oil Field, China). The influence of that microorganism on the phenanthrene and pyrene degradation was studied. For both PAHs, four different initial concentrations, 10, 50, 100, and 200 mg/kg, were used. It was demonstrated that when higher initial concentrations were used, of 100 and 200 mg/kg, the phenanthrene degradation was higher, at the level of 83.7 and 70%, respectively (after 350 h), than when its initial levels were lower. For pyrene, biodegradation was the highest, of 74.6%, for the initial pyrene level of 100 mg/kg, and the lowest, of 32.1%, when it was at the level of 200 mg/kg [73].

Barnes et al. investigated the phenanthrene degradation using ten fungal cultures isolated from the aquatic environment: P. citrinum, A. sclerotigenum, A. polyporicola, A. versicolor, F. equiseti, Fusarium sp., Aspergillus sp., A. favus, and A. sydowii. Among the ten isolates studied, the degradation ranged from 22.1 to 100% after 23 days of incubation. The highest degradation level was achieved with F. equiseti [64].

The microbial degradation of the phenanthrene by screened fungi found in the natural environment was conducted, to select fungi for the phenanthrene bioremediation. The highest degradation level, of 72%, was obtained when Trichoderma sp. S019 was incubated for 30 days after 0.1 mM of phenanthrene were added to the liquid medium, while it reached 31% when 1 mM of the studied PAH was added [74].

3.1.5 Pyrene

On Day 4 of the study, the highest pyrene concentration amounted to 0.05 mg/kg, and the lowest amounted to 0.019 mg/kg. On Day 14 of the study, pyrene reached its highest level of 0.011 mg/kg in the control samples, and the lowest level, of 0.006 mg/kg, in the samples treated with Formulation 5. On the last day of the study, pyrene was at the highest level, of 0.006 mg/kg, in the control samples, and at the lowest concentration of 0.003 mg/kg in the samples treated with Formulations 3, 4, 5, and 6 (Figure S13).

It was observed for pyrene that all studied EM formulations accelerated its degradation, which ranged between 85.9 and 91.8%. The degradation was the highest after treatment with Formulation 1, and the lowest for Formulation 6 (Table 3).

The studies on the influence of the immobilized (a hybrid carrier consisting of polyvinyl alcohol with sodium alginate and activated carbon) fungi, Aspergillus niger, Trichoderma sp., and Fusarium sp., on the pyrene degradation were conducted by Wang et al. The initial pyrene concentration was 100 mg/kg. After 240 h of incubation, the pyrene degradation amounted to 63% for Trichoderma sp., 49% for A. niger, and 69% for Fusarium sp. In the case of the synergistic effect of A. niger and Fusarium sp., the highest pyrene degradation of 81% was recorded [75].

A strain of the bacterium Achromobacter xylosoxidans PY4 was isolated from a polluted area (Jubail, Saudi Arabia) and characterized. It was demonstrated that this bacterium is able to metabolize and use pyrene as its only source of carbon. The PY4 strain was capable of decomposing 80% of pyrene at the initial level of 100 mg/L in 25 days [76].

Barnes et al. analyzed the pyrene degradation using ten fungal cultures, isolated from the aquatic environment, of P. citrinum, A. sclerotigenum, A. polyporicola, A. versicolor, F. equiseti, Fusarium sp., Aspergillus sp., A. favus, and A. sydowii. Among the ten isolates studied, the degradation ranged from 45.7 to 100% after 23 days of incubation. The maximum degradation level was observed when F. equiseti and P. citrinum were used [64].

The APC5 strain of Coriolopsis byrsina, white rot fungi, was isolated in the Surguja district of Chhattisgarh, India, and used in the studies on the pyrene biodegradation. The maximum degradation reached the level of 96.1%. C. byrsina produced a significant amount of ligninolytic enzyme in the mineral salt broth (MSB) containing pyrene [77].

Hadibarata et al. studied the pyrene degradation by Candida sp. S1, the yeast isolated from the tropical rain forest. Biodegradation was at the level of 35% after 15 days, but the percentage of pyrene decomposition increased up to 75% with 24 g/L of sodium chloride added, and decreased as the salinity increased. Under the acidic conditions, biodegradation increased up to 60% at the pH of 5. It was also found that higher glucose concentrations, exceeding 10 g/L, had no significant effect on the pyrene biodegradation, while agitation proved to have greater influence [31].

3.1.6 Chrysene

On Day 4 of the study, the highest chrysene concentration amounted to 0.043 mg/kg, and the lowest amounted to 0.021 mg/kg. On Day 14, the substance was at the maximum concentration of 0.012 mg/kg in the samples treated with Formulation 7, and at the lowest, of 0.004 mg/kg, in the samples containing Formulation 5. On Day 35 of the study, chrysene level was the highest, of 0.008 mg/kg, in the control samples, and the lowest, of 0.004 mg/kg, in the samples treated with Formulations 4, 5, and 6 (Figure S8).

The use of all EM formulations accelerated the chrysene decomposition. The highest degradation, of 85.5%, was obtained for Formulation 1, while it was the lowest, of 80.3%, for Formulation 5 (yeast strains Y. lipolytica) (it accelerated the chrysene decomposition by 9.6% versus the control) (Table 3).

A fungus, Polyporus sp. S133, isolated from the soil polluted with crude oil, was used in the studies on the chrysene biodegradation in a culture without and with a synthetic surfactant (Tween 80). The maximum degradation intensity, of 86%, was achieved for the fungus incubated with 0.5% Tween 80 solution for 30 days. When the microorganism was incubated without that surfactant, the degradation was only at a level of 30% [78].

Similar studies were conducted in a liquid MSB. The maximum rate of the chrysene degradation, of 65%, was achieved when Polyporus sp. S133 was used with an addition of polypeptone as a source of vitamins, carbon, and amino acids for the microorganisms, when compared to the 24% degradation in the pure MSB medium [79].

Other research focused on the influence of a bacterial consortium on the chrysene degradation in the soil polluted with crude oil. The consortium consisting of Bacillus cereus and Pseudomonas putida 10 and 15% bacteria with ratios – 2:3, 1:1, 3:2 was added into a slurry bioreactor. The initial chrysene concentration amounted to 24.48 ng/µL. After 49 days of the experiment, the chrysene degradation was at the level 67.01, 69.1, and 64.54%, in the case of the 10% bacterial consortium, and of 89.39, 93.58, and 91.73%, for the 15% bacterial consortium [80].

In yet another study, Vaidya et al. developed a bacterial consortium consisting of Rhodococcus sp., ASDC1; Bacillus sp. ASDC2; and Burkholderia sp. to study the chrysene degradation. Chrysene was utilized by the consortium as a sole source of carbon and energy, with the maximum degradation rate of 1.5 mg/L/day and the maximum growth rate of 0.125/h, under optimized conditions of the pH of 7.0, the temperature of 37°C under aeration of 150 rpm, on gyrating shaking. The maximum degradation of 96% was obtained in the polluted, non-sterile soil sediment [81].

3.1.7 Benzo[a]anthracene

On Day 4, the benzo[a]anthracene level in the soil samples was between 0.016 and 0.041 mg/kg. On Day 14 of the experiment, the maximum concentration was noted in the control samples and following treatment with Formulation 1, amounting to 0.008 mg/kg, while it was the lowest, of 0.004 mg/kg, after treatment with Formulations 3, 4, and 5. On the last day of the study, the benzo[a]anthracene level was the highest in the control samples and amounted to 0.005 mg/kg, while it was the lowest after treatment with Formulations 4, 5, and 6, and amounted to 0.002 mg/kg (Figure S3).

The benzo[a]anthracene decomposition was accelerated by the use of all formulations. It was the highest, of 90.8%, after treatment with Formulation 3, and the lowest, of 88.2%, with Formulation 7 (Table 3).

The potential of benzo[a]anthracene biodegradation by Panebacillus sp. strain HD1PAH, a new strain isolated from the soil polluted with crude oil, was studied by Deka and Lahkar. The study was conducted in laboratory conditions for 144 h, and samples were collected seven times. Benzo[a]anthracene was the only source of carbon, at the level of 10 mg/L in the MSM. The maximum degradation of that PAH by the Panebacillus sp. strain HD1PAH was 82.01% during 144 h of incubation [82].

Rocha et al. also analyzed the P. ostreatus influence on the benzo[a]anthracene degradation in the soil. P. ostreatus degraded about 90% of this compound in the soil enriched at the level of 30 mg/kg, and 80% of benzo[a]anthracene when the soil was enriched at the level of 60 mg/kg after a period of incubation of 15 days [63].

Barnes et al. analyzed the degradation of benzo[a]anthracene using ten fungal cultures, isolated from the aquatic environment: P. citrinum, A. sclerotigenum, A. polyporicola, A.versicolor, F. equiseti, Fusarium sp., Aspergillus sp., A. favus, and A. sydowii. Among the ten isolates studied, nine isolates achieved a 100% removal of benzo[a]anthracene after incubation of 23 days [64].

Wu et al. studied the degradation of benzo[a]anthracene by fungi isolated from the environment of PAH-contaminated mangrove sediments in Ma Wan, Hong Kong. Benzo[a]anthracene at the concentration of 20 mg/L was added to the MSM for initial screening of PAH-degrading fungi; and finally, two fungal species capable of using the studied PAH as the sole source of carbon were isolated and identified as F. solani – MAS2 and MBS1 strains. Benzo[a]anthracene removal reached 60% of the added amount after 40 days of incubation in the samples containing the MBS1 strain [71].

3.1.8 Benzo[a]pyrene

On Day 4 of the study, the benzo[a]pyrene level was the highest of 0.039 mg/kg, and the lowest of 0.015 mg/kg. On Day 14 of the study, the compound concentration was the highest in the control samples and those with Formulation 1 (0.01 mg/kg), and the lowest in the samples spiked with Formulation 5 (0.004 mg/kg). On the last day of the study, the benzo[a]pyrene concentration was the highest, of 0.007 mg/kg, in the control samples, while it is at the minimum level of 0.003 mg/kg in the samples treated with Formulations 4, 5, 6, and 7 (Figure S4).

The use of all EM formulations accelerated the degradation of benzo[a]pyrene, when compared to the control, and it ranged between 82.0 and 87.3%. Formulation 1, containing bacteria from Bifidobacterium, Bacillus, Lactococcus, Lactobacillus, Rhodopseudomonas, and Streptococcus genera, and yeasts S. cerevisiae, most strongly accelerated the decomposition of this substance (it accelerated the benzo[a]pyrene degradation by 9.8% versus the control) (Table 3).

Su et al. studied the influence of two microorganisms, Bacillus sp. SB02 and Mucor sp. SF06, on the benzo[a]pyrene degradation in the soil. Both SF06 and SB02 were isolated from the soil collected from the Shenfu Irrigation Area, China. The researchers studied and compared characteristics of the benzo[a]pyrene degradation, by free and by co-immobilized microorganisms. The level of the benzo[a]pyrene degradation was verified five times, on Days 7, 14, 21, 28, and 42. A higher degradation of that compound was achieved for microorganisms in the co-immobilized system. The highest degradation level was 79.6% for free microorganisms, and 95.3% for co-immobilized microorganisms [83].

Su et al. conducted similar studies on the benzo[a]pyrene degradation in the soil by the fungus, Mucor sp., free and immobilized. This microorganism was isolated from the soil contaminated with PAHs from the Shenfu Irrigation Area. The initial concentration of the studied PAH was 50 mg/kg. The degradation rate was higher for the immobilized fungi. The maximum degradation amounted to 68% for the immobilized fungi and 52% for free microorganisms on Day 42 of the experiment [84].

The influence of two bacteria, Bacillus flexus S1I26 and Paenibacillus sp. S1I8, isolated from the soil polluted with crude oil was studied. It was demonstrated that both isolates were able to secrete biosurfactant that increased benzo[a]pyrene solubility. On Day 21 of the experiment, B. flexus S1I26 degraded 70.7% of benzo[a]pyrene, while Paenibacillus sp. S1I8 achieved a higher degradation level, amounting to 76.8%. The results suggest that isolates producing biosurfactants can be a potential tool for PAHs biodegradation in the soil and they can be used to bioremediate sites polluted with those compounds [85].

The studies concerning the influence of fungi, Trichoderma sp., Fusarium sp., and A. niger, on the benzo[a]pyrene degradation were conducted. A hybrid carrier consisting of polyvinyl alcohol, sodium alginate, and activated carbon was used to immobilize the microorganisms. The initial concentration of benzo[a]pyrene was 100 mg/kg. After 240 h of incubation, the benzo[a]pyrene degradation amounted to 34% for Trichoderma sp., 29% for A. niger, and 37% for Fusarium sp. The degradation levels significantly exceeded those obtained for free fungi. Additionally, the synergistic effect of those microorganisms was verified, and for benzo[a]pyrene the highest degradation was obtained with A. niger and Fusarium sp., amounting to 43% [75].

Mandal et al. analyzed the benzo[a]pyrene degradation by a yeast consortium including Rhodotorula sp. NS01, H. opuntiae NS02, D. hansenii NS03, and H. valbyensis NS04, isolated from the contaminated soil. The maximum degradation amounted to 76% after 6 days in the aqueous medium under optimized conditions of pH 7.0, temperature of 30°C, shaking speed of 130 rpm, an inoculum dose of 3% (w/v), and the initial benzo[a]pyrene concentration of 50 mg/L [35].

3.1.9 Benzo[b]fluoranthene

On Day 4, the benzo[b]fluoranthene concentration was in the range from 0.013 to 0.039 mg/kg. On Day 14 of the study, the benzo[b]fluoranthene concentration was the highest in the control samples and amounted to 0.01 mg/kg, while it was the lowest in those with Formulation 5, amounting to 0.004 mg/kg. On the last day of the study, the studied substance was at the highest level, of 0.006 mg/kg, in the control samples, and at the minimum level of 0.003 mg/kg in the samples treated with Formulations 3, 4, 5, 6, and 7 (Figure S5).

The benzo[b]fluoranthene degradation was higher following treatment with EM formulations, excluding Formulation 6 (the yeast D. hansenii and its metabolites, bacteria from Bacillus genus), for which the degradation was similar to the control, at a level of 78.5%. The highest degradation, of 88.4%, was achieved for Formulation 1 (it accelerated the benzo[b]fluoranthene decomposition by 9.4% versus the control) (Table 3).

Kumari et al. demonstrated the ability of Ochrobactrum anthropi, S. maltophilia, P. mendocina, P. aeruginosa, and Microbacterium esteraromaticum to biodegrade several PAHs, including benzo[b]fluoranthene, during the bacteria exposure to crude oil. The benzo[b]fluoranthene concentration in the sample of oil collected from the Digboi refinery in India amounted to 6.5 mg/L. The experiments were conducted in laboratory conditions. The samples were tested on Days 15, 30, and 45. The degradation was the lowest, at the level of 34.8%, for P. mendocina, and the highest, of 61.2%, for P. aeruginosa. However, the consortium of the described bacteria achieved the enhanced benzo(b)fluoranthene degradation of 72.8% [86].

Treviño-Trejo et al. isolated and selected bacteria able to degrade benzo[b]fluoranthene. The isolates ability to tolerate concentrations of 50 and 75 mg/L of the studied PAH in the liquid medium was evaluated. The selected isolates were identified as belonging to Bacillus, Gordonia, Pseudomonas, Rhodococcus, Ochrobactrum, and Amycolatopsis genera. All isolates tolerated and grew at the benzo[b]fluoranthene concentrations tested. The most prominent was Amycolatopsis sp. Ver12, which removed 47% of benzo[b]fluoranthene; furthermore, with the addition of the yeast extract, it removed 59% of the studied compound [87].

3.1.10 Benzo[k]fluoranthene

On Day 4 of the study, the highest benzo[k]fluoranthene concentration amounted to 0.043 mg/kg, and the lowest amounted to 0.013 mg/kg. On Day 14 of the study, the concentration was the at the highest level, of 0.011 mg/kg, for the control samples and the samples treated with Formulation 1, and the lowest, of 0.004 mg/kg, following treatment with Formulation 5. On the last, 35th day of the study, the studied substance was at the highest level, of 0.008 mg/kg, in the control samples, and at the lowest level, of 0.003 mg/kg, in the samples treated with Formulations 4, 5, 6, and 7 (Figure S6).

The use of all EM formulations accelerated the benzo[k]fluoranthene decomposition. The degradation was the lowest for Formulation 6 (75.5%), and the highest for Formulation 1 (86.6%) (it accelerated the benzo[k]fluoranthene decomposition by 11.7% versus the control) (Table 3).

The results for benzo[b]fluoranthene and benzo[k]fluoranthene are very similar, due to the similar structure of these two PAHs.

Arulazhagan et al. studied the benzo(k)fluoranthene degradation by S. maltophilia strain AJH1, bacteria isolated from soil samples collected from different areas owned by the Saudi Arabian Mining Company. Strain AJH1 was able to degrade the benzo(k)fluoranthene at the concentration of 10 mg/L in the acidophilic MSM at the pH of 2. The maximum degradation level was 79% after 11 days [88].

Maeda et al. reported the studies on the benzo(k)fluoranthene degradation by the soil bacterial isolates Sphingobium sp. strain KK22. Benzo(k)fluoranthene concentration was reduced by 70% in 20 days of the experiment [89].

3.1.11 Dibenzo[a,h]anthracene

On Day 4 of the study, the highest dibenzo[a,h]anthracene concentration amounted to 0.041 mg/kg, and the lowest amounted to 0.014 mg/kg. On Day 14 of the experiment, the highest dibenzo[a,h]anthracene concentration, of 0.013 mg/kg, was found after treatment with Formulation 1. The lowest level, of 0.006 mg/kg, was noted in the samples treated with Formulations 5, 6, and 7. On Day 35 of the study, the substance was at its highest level, of 0.006 mg/kg, in the control samples, while it was at the lowest concentration, of 0.003 mg/kg, in the samples treated with Formulations 4, 5, 6, and 7 (Figure S9).

The use of EM formulations accelerated the dibenzo[a,h]anthracene degradation when compared to the control, except for Formulation 6 (77.5%). The highest degradation, of 87.9%, was achieved with Formulation 1 (Table 3).

The influence of eight fungal species A. praecox, Agrocybe dura, Kuehneromyces mutabilis, Hypholoma capnoides, S. rugosoannulata, Stropharia coronilla, S. cubensis, and S. hornemannii, on the dibenzo[a,h]anthracene degradation was studied by Steffen et al. The highest degradation of that compound, of 84%, was obtained for S. coronilla [90].

Dibenzo(a,h)anthracene biodegradation by indigenous strains of aerobic heterotrophic bacteria and cyanobacteria isolated from the Bodo creek, a moderately salty aquatic site polluted with crude oil, was monitored. Dibenzo(a,h)anthracene levels were reduced to 0 mg/L in all treatment options on Day 56 [91].

3.1.12 Benzo[ghi]perylene

Benzo[ghi]perylene was yet another studied substance. On Day 4, this substance was at the highest concentration of 0.052 mg/kg, and at the lowest level of 0.020 mg/kg. On Day 14 of the study, the benzo[ghi]perylene concentration was the highest in the control samples and amounted to 0.019 mg/kg, while it was the lowest in the samples spiked with Formulations 5 and 7, amounting to 0.011 mg/kg. On Day 35, the concentration was the highest, of 0.008 mg/kg, in the control samples, and the lowest, of 0.004 mg/kg, in the samples treated with Formulation 6 (Figure S7).

After treatment with Formulations 5 and 6, the benzo[ghi]perylene degradation was comparable to the control (ca. 79%), and for Formulation 7 the degradation was lower, of 76.6%. The highest degradation was obtained for Formulation 1, of 86.3% (Table 3).

Studies were conducted on the benzo[ghi]perylene biodegradation by B. licheniformis STK 01, B. subtilis STK 02, and P. aeruginosa STK 03, which were isolated from wood chips, coal tar, and an oil spill site. In the experiment, mono-septic cultures or co- and augmented cultures were used. The benzo[ghi]perylene concentration was 25 mg/kg of the soil. After 60 days of the experiment, 52.73, 40.50 and 58.42% of benzo[ghi]perylene were biodegraded by B. licheniformis, B. subtilis, and P. aeruginosa, respectively. The highest rate of the benzo[ghi]perylene degradation, of 60.76%, was reached with B. licheniformis and B. subtilis [72].

Mandal et al. studied the benzo[ghi]perylene degradation by the yeast consortium YC04, consisting of three isolates – Rhodotorula sp. NS01, D. hansenii NS03, and H. valbyensis NS04 in the MSM. The YC04 efficiency in benzo[ghi]perylene remediation was tested in a presence of ZnO nanoparticles and produced a biosurfactant in the growth medium. The maximum benzo[ghi]perylene biodegradation was found to be 63.83% at central values of all factors, a pH of 7.0, at a temperature of 30°C, and at a shaking speed of 130 rpm in the presence of 2 g/L of ZnO nanoparticles, using 3% inoculum doses of the yeast consortium YC04 after 6 days of incubation [34].

3.1.13 Indeno[1,2,3-cd] pyrene

On Day 4, the highest determined indeno[1,2,3-cd] pyrene level was 0.035 mg/kg, while the lowest concentration amounted to 0.015 mg/kg. On Day 14, the highest level of the studied substance was 0.031 mg/kg in the control samples, while the lowest concentration was found in the samples treated with Formulation 7 (0.014 mg/kg). On the last day of the experiment, the highest indeno[1,2,3-cd]pyrene level of 0.008 mg/kg was found in the control samples, while its level was the lowest (0.002 mg/kg) following application of Formulation 6 (Figure S11).

The use of EM formulations significantly accelerated the indeno[1,2,3-cd]pyrene degradation versus the control. The highest degradation, of 87%, was obtained for Formulation 5, while it was the lowest, of 83.3%, for Formulation 7 (it accelerated the indeno[1,2,3-cd]pyrene decomposition by 17% versus the control) (Table 3).

Two bacterial strains, Acinetobacter baumannii INP1 and Pseudomonas taiwanensis PYR1, were isolated from Liaohe, China, contaminated with PAHs. These microorganisms were able to degrade 55.3% of indeno[1,2,3-cd]pyrene after a period of 30 days. These strains’ ability to degrade pyrene was also studied, and it was found that they degraded 58.2% of pyrene [92].

Barnes et al. analyzed the degradation of indeno[1,2,3-cd]pyrene using ten fungal cultures, isolated from the aquatic environment – P. citrinum, A. sclerotigenum, A. polyporicola, A.versicolor, F. equiseti, Fusarium sp., Aspergillus sp., A. favus, and A. sydowii. All tested isolates degraded almost 100% of indeno[1,2,3-cd]pyrene [64].