Petroleum is a non-renewable resource of economic importance for several countries [1] , including Ecuador. However, crude oil exploitation is a highly contaminant industry. Water and soil pollution might occur in any stage of production. In a highly biodiverse country, as Ecuador, the impact on habitats for local flora and fauna is concerning, in addition to the effects on people’s health [2] [3].

Petroleum hydrocarbons (PHs) are categorized into four broad classes of chemical compounds, namely, the aliphatic, aromatic, resin-based, and asphaltene-based hydrocarbons. Each group possesses different physicochemical characteristics and susceptibility to degradation [4] [5].

All hydrocarbon compounds derived from petroleum sources are generally described as total petroleum hydrocarbons (TPHs) and are categorized as aromatic, aliphatic, resin-based and asphaltene-based hydrocarbons. Aliphatic hydrocarbons correspond to linear or cyclic hydrocarbon chains of variable size and structure. It is widely studied for their uses as fuels and solvents. Aromatic hydrocarbons have a variable structure, size and complexity and range from monocyclic structure compounds to compounds formed by long and complex chains, constituted by aromatic rings [6]. Polycyclic aromatic hydrocarbons have been mostly studied, because some of them constitute a wide variety of products of biological, chemical and industrial importance [6]. However, they are also among the pollutants with the greatest biological impact due to their carcinogenic and mutagenic effects [7] [8] [9]. Degradation of these different compounds is highly related to their physical chemical characteristics [10] [11] [12].

Microbial groups, such as bacteria, yeast, and fungi have been identified as principal agents in the degradation of PHs, even though their degradation efficiencies are varying. However, bacteria are the most active and primary degraders of spilled oil in the environment [13]. Remediation techniques have been developed using microorganisms, such as bacteria, yeast and fungi, with varying degradation efficiencies [14]. It is generally suggested that these microorganisms have the metabolic capacity to use hydrocarbons as a carbon source [15]. Also phytoremediation is considered as cost-efficient and eco-friendly and can be implemented after an initial degradation either by bioremediation or physical, chemical and thermal processes.

Bioremediation of petroleum hydrocarbons using native bacterial and fungal isolates from the Ecuadorian soil microbiome has been previously reported [14]. Generally, bioremediation techniques rely on bacteria given their rapid reproduction cycles, their voracity and the facilities for the study of their metabolic pathways [16] [17]. However, recently various studies have reported fungal organisms that exceed bacterial capacities in hydrocarbon biodegradation [18]. Fungi are used due to their particular metabolic capacities, which mediate the transformation of a wide variety of organic compounds to less complex compounds that are later incorporated into their metabolisms [19] [20] ; these processes are facilitated due to the characteristics of their complex enzymatic system and their vigorous hyphae growth [15] [19] [21]. Thus, they offer an undeniable potential for their use in bioremediation processes, accordingly, the study of the diversity and identification of species and fungal strains of biotechnological utility should be a priority in an avid field of constant innovation such as environmental bioremediation.

In recent years, endophytic fungi have generated major attention in the scientific community due to their wide diversity and particular metabolic capacities [22]. These microorganisms colonize plant tissues, without causing damage or apparent symptoms of disease in their host [13] [23] [24]. They are located in the intercellular spaces and the symplast [25]. Plant and fungi coexist, establishing between them a mutualistic interaction, dependent on the virulence of the fungus and the defenses of the plant. Both elements are influenced by environmental factors and the developmental stage of each organism [24] [26] [27].

Plant-endophyte interaction, be it with bacterial or fungal endophytes, generally improve plant adaptation to the environment by increasing tolerance to biotic and abiotic stress, changes in temperature and salinity, development of resistance to diseases, herbivory, insects, nematodes, bacteria and other pathogenic fungi [28]. The development of resistance to normally lethal or highly hazardous pollutants, such as crude oil, occurs when plants are able to metabolize harmful compounds by extracellular enzymes secreted by endophytic fungi, this process could be developed inside the plant or at soil level by enzymatic secretion through the root [29].

In Ecuador, main oil reservoirs are found in the Cretaceous Hollin and Napo geological formations in the Oriente basin. The Oriente basin, which covers about 100,000 km², lies between the Andes on the west and the Guyana shield on the east. The basin extends northward into the Putumayo basin in Colombia, and southward into the Maranon basin in Peru [30]. The principal reservoir, the Lower Cretaceous Hollin Formation is characterized by structurally controlled oil accumulations found in the Cretaceous sandstones of this formation. Geochemical analyses indicate that the oil migrated into these structures from Cretaceous source rocks in the eastern Cordillera and southernmost Oriente basin [31]. The oil is trapped in structures of Cretaceous-Oligocene age [32]. On the Hollin-Loreto roadway that connects Ecuador’s capital city, Quito, to the Ecuadorian Amazon basin, outcrops can be observed with these oil accumulations. Various plant species have colonized, adapted and proliferated successfully in habitats contaminated with petroleum [33] , with this consideration in mind, the vegetation found on the outcrops on the Hollin-Loreto roadway most probably exhibit similar adaptive features.

Therefore, the exploration and identification of the endophytic fungal diversity these plants harbor could lead to the discovery of fungi with metabolic capacities to biodegrade petroleum hydrocarbons by using these as a carbon source, facilitating the adaptation and host plants survival to adverse conditions. These findings could contribute to the innovation and development of new technologies for environmental bioremediation.

This study is primarily aimed to describe plant fungal endophytic diversity of plant communities in a tropical lowland ecosystem exhibiting long term petroleum contamination, as well as to evaluate petroleum hydrocarbon biodegradation capacities of the endophytic fungi isolated from plants adapted to crude oil contaminated habitats.

Plant adaptation to highly contaminated habitats is restricted to a handful of species that tolerate such conditions [44]. Evidence has shown that endophytes contribute to plant adaptation to stress conditions, stimulating growth, productivity, carbon sequestration and tolerance to contaminants [45].

Deng and Cao [45] suggested that the degree of substrate contamination by hydrocarbons negatively affect plant species diversity and abundance. However, the sampling site of this study exhibits geological oil outcrops with typical Amazonian tropical humid forest vegetation developing in situ, turning this area into a point of interest for the diversity study and capabilities of endophytic fungi from plants adapted to this type of habitat.

The endophytic fungi isolates were subjected to in vitro tests to evaluate their hydrocarbon biodegradation capacity [18]. The qualitative essays showed the hydrocarbon biodegradation potential of 53 endophytic fungi, as indicated in Table 2. The essays are based on the fact that during microorganisms’ oxidation reactions, electrons are transferred to an electron acceptor such as oxygen (O₂), nitrates ( NO 3 − ) or sulfates ( SO 4 2 − ) [46] [47]. By incorporating an electron accepting reagent into the culture medium, such as DCPIP, it is possible to demonstrate the organisms’ capacities to use petroleum hydrocarbons as a carbon source through the DCPIP color change, from blue (oxidized) to colorless (reduced) [48]. This redox technique demonstrates that 53 fungal isolates possess the ability to degrade crude oil. Within these 53 endophytes, a subgroup composed of 10 individuals presented outstanding results in the qualitative essays, clarifying completely the culture medium in less time than established for the essay (<10 days) (Figure 1).

With exception of plant families Orchidaceae sp. and Poaceae sp., all the collected plants have fungal organisms with variable capacities to degrade hydrocarbons in this essay. Evidence obtained by [49] and [50] demonstrates the plant endophytic composition could be affected by contaminants, stimulating the proliferation of organisms tolerant to these conditions. Interestingly, the plants with the highest number of endophytic isolates, Marattia sp. (Marattiaceae) and FM006 (unidentified), with 12 and 10 isolates respectively, correspond to plants with the highest number of fungi with hydrocarbon biodegradation capacities (Table 2), this results support the hypothesis that endophytes contribute to a better adaptation of the host plant to contaminated substrates [28].

Twenty-three plant samples were collected in the site (Table 1), including stem and leaves samples. The families Melastomataceae, Boraginaceae and Poaceae stood out for their predominance in the area, representing up to 70% of the species present in the area.

To a lesser degree, individuals from the families Rubiaceae, Gunneraceae and Urticaceae were found. A previous study [33] reported several families in common with this study (Melastomataceae, Boraginaceae, Poaceae, Euphorbiaceae, Bignoniaceae and Rubiaceae) as common species in the tropical Ecuadorian rainforest but evidenced an abnormally higher abundance of them in contaminated secondary forest in contrast to unpolluted secondary forest and pristine forest [33]. In our study the predominant families were Melastomataceae, Rubiaceae, Fabaceae and Euphorbiaceae. The marked predominance of the individuals of the family Melastomataceae could demonstrate a relationship between the family and the colonization of this type of habitat [33]. Likewise, the highest number of fungal endophytes were isolated from plants species of the Melastomataceae, Marattiaceae, Polypodiaceae and Euphorbiaceae which may indicate a strong interaction between the plant and the fungal endophytes, similar to bacteria that are in intimate association with plants which are known to consume alkanes and aromatic hydrocarbon exudates granting them the capacity to in degrade organic contaminants [51].

A total of 133 endophytic fungi were isolated from stem and leaf tissues of 23 plants species (Table 1). These figures are congruent with the suggestion that Ecuadorian endophytes are hyperdiverse [52]. The taxonomic positions of 53 fungi were established through a phylogenetic analysis (Figure 3). The phylogenetic tree shows the four phyla in the fungi kingdom, Ascomycota, Basidiomycota, Zygomycota and Spizellomyces punctatus (phylum Chytridiomycota) as outgroup. The analysis shows that most of the isolated ascomycetes constitute the orders Diaporthales, Xylariales and Glomerellales.

Within the order Diaporthales 14 fungi isolates belonging to three families were identified: Diaporthaceae, Valsaceae and Schizoparmaceae. Among the 14 isolates of the order Diaporthales, the endophyte Phomopsis sp. 1 (Table 3) showed a hydrocarbon removal rate of 90.7%. Biodegradation of phenanthrene and 4-hydroxybenzoic acid has been reported by fungi from the genus Phomopsis [53] [54] ; however, this is the first report of crude oil biodegradation for fungi from the genus.

Within the Xylariales order, 12 fungi were identified, 11 belonging to the family Xylariaceae and 1 identified until order level (Table 5). Among the 12 individuals, the endophyte Xylaria sp. 1 showed a hydrocarbons removal rate of 98.8% (Table 3). Pyrene and benzopyrene biodegradation has been reported by Xylaria regalis [55] but there are no reports to crude oil biodegradation.

The order Glomerellales groups 8 fungi, all belonging to the family Glomerellaceae and the genus Colletotrichum (Table 5). Within this order, three individuals with hydrocarbon removal rates ranging from 70.4% to 97.5% (Table 3). Colletotrichum gleosporoides is known to produce extracellular lipases [56] , enzymes that have been widely studied for their ability to degrade hydrocarbons [57] [58].

However, when submitting Colletotrichum gleosporoides to hydrocarbon biodegradation essay the lipase production capacity was inhibited, possibly due to the impact of the toxic experimental conditions [58].

The order Hypocreales has three identified isolates; the hydrocarbon removal rate to Clonostachys sp. 1 was 31.5% and 97.4% to Clonostachys sp. 2 (Table 3). There are no previous reports about hydrocarbon biodegradation by individuals from the genus Clonostachys. But, metabolic capacities studies with individuals from the genus Clonostachys determine zearalenones biodegradation [59].

Aspergillus sp. showed a removal rate of 97.6% (Table 3). Previous studies conducted with free-living fungi reported the capacities of Aspergillus fumigatus and Penicillium chrysogenum for the production of laccases [58] , family of enzymes that are actively involved in the hydrocarbons biodegradation processes by oxidizing them [57] [58]. Additionally, there are biodegradation reports of phenanthrene, pyrene and benzopyrene by Aspergillus niger [60]. Biodegradation of fluoranthene by Penicillium sp., Pyrene by Penicilliumjanczewskii and P. janthinellum and crude oil by Penicillium sp. [61] [62]. These works results were obtained with free-living fungi but, belong to the same genus of the endophytes of our study, thus believe the enzymes involved in hydrocarbon biodegradation processes could be the same ones already reported.

The order Pleosporales is represented by four fungi, three belong to the genus Saccharicola, family Lophiostomataceae (Table 5). The endophyte Saccharicola sp. 1 showeda hydrocarbon removal rate of 78.9% (Table 3). This is the first report of hydrocarbon biodegradation by a fungus from the genus Saccharicola (97% ID). However, the production of extracellular protease enzymes has been confirmed in individuals of the genus [63] , additional studies have identified fungal proteases due to their hydrolytic activity could participate in hydrocarbon degradation processes [57] [58].

Quantification of TPH by IR is based on the measurement of the absorbed energy to produce stretching and bending vibrations of the molecules. The different functional groups and bond types absorb radiation at different frequencies and intensities. The absorption intensity is directly proportional to the bonds number. This is correlated to the hydrocarbons concentration present in the sample [64]. These results were compared with a control sample to establish the hydrocarbon removal rates.

Previous studies with the same hydrocarbon biodegradation methodology using free-living mushrooms, reported removal rates ranges from 14.9% to 43.4% [35]. In this study we obtained variable degradation rates, from 31.5% (minimum), to 99.6% (maximum) of hydrocarbon removal, as shown in Table 3.

The analysis also determined a high variation (degradation capacity) among the tested endophytes. The replicates of the trials showed no statistically significant differences.

Additionally, Tukey multiple comparisons analysis grouped the endophytes that showed statistically homogeneous performances among themselves, establishing 3 subgroups, the first subgroup agglutinates the five endophytes with the best performances (Xylaria sp. 1, Clonostachys sp. 2, Colletotrichum sp. 1, Verticillium sp. and Aspergillus sp.) (Table 6). In order to corroborate the results obtained with the best five endophytes, a new analysis was carried out, quantifying the hydrocarbon present in the culture medium by GC. In this TPH quantification method, a mobile phase (inert gas N₂) and a stationary phase (capillary column) are used to separate the hydrocarbon mixture from the samples in their individual components, carried by the mobile phase through the stationary phase. The separation is carried out by a combination of factors including the boiling point, polarity and affinity differences between the different hydrocarbons and the column, making the concentrations estimation had a greater accuracy and allows establish differences between the quantification methods used.

Removal rates may be affected by several factors, including the test temperature. In the present study, the tests were carried out at a temperature of 23˚C in absence of an incubator with agitation, but evidence shows that the optimum temperature for carrying out this type of essays ranges between 28˚C and 31˚C [65] [66] , suggesting the results could be improved modifying the experimental temperature.

Another factor was the addition of a surfactant to the culture medium. There are studies where the culture medium was not supplemented with a surfactant to emulsify the mixture between crude oil and water, making the biodegradation process even more complex for the evaluated microorganisms [67]. In this case, Tween^Ò80, the surfactant used in this study, is nontoxic and a stimulant of manganese peroxidase production [68] ; the results obtained indicate that it was the appropriate reagent to optimize the enzymatic activity without toxic effects.

The crude concentration evaluated in this study was 1%; further studies could evaluate the performance of these endophytic fungi at higher concentrations, the optimum concentrations for fungi range between 1% and 5% [69]. The crude oil concentration used is determinant to obtain optimal results. Evidence provided by [70] shows that the degradation percentage of a bacterial consortium culture decreased from an initial 78% to 52% by increasing the crude oil concentration from 1% to 10%.

The results shown in Table 3 demonstrate the enormous potential of isolated fungi to eventual uses in the bioremediation field. Six fungi (Xylaria sp. 1, Clonostachys sp. 2, Colletotrichum sp. 1, Verticillium sp., Aspergillus sp. and Mucoraceae sp.) showed removal rates higher than 90%, under the tested conditions, are highly encouraging results for a fungal assay and even more in the case of endophytic fungi.

Reference [50] isolated endophytic bacteria from roots of plants that growing in a contaminated with crude oil environment and evaluated the capacities to biodegrade crude by bacterial isolates at 1% concentration; obtaining similar hydrocarbon removal rates to those obtained in this study. The isolated Streptomyces sp. HIh9 showed a 97.5% hydrocarbon removal rate after 7 days of incubation, while in our study the endophyte fungus Verticillium sp. showed 99.64% after 30 days of incubation, which evidences that plants harbor endophytic contaminant degrading organisms as adaptation mechanism to hostile habitats, due to the plant alone is unable to metabolize these compounds. This study demonstrates the potential for using fungal endophytes in biodegradation of crude oil, either through biotechnological applications or by improving phytoremediation strategies aiding plants to tolerate contaminant-induced stress, especially in countries with economies based on fossil fuel exploitation.

Petroleum hydrocarbon biodegradation is a complex process, depending on the crude oil nature, composition, environmental conditions and soil microbial community. The presence of hydrocarbon-degrading microorganisms is an adaptive response to the environmental contamination conditions [71]. Adaptation mechanisms include selective enrichment and genetic changes that stimulate the complex hydrocarbon biodegradation. The plants forced to develop in this environment stimulate the growth of hydrocarbon-degrading microorganisms to detoxify the plant from substances that normally could not be processed by itself. Fifty-three hydrocarbon-degrading endophytic fungi were isolated from plants growing in crude oil contaminated soils at Ecuadorian Amazon. We identified six endophytic fungi that showed Total Petroleum Hydrocarbon removal rates higher than 90% after 30 days of incubation. The endophyte Xylaria sp. 1 showed the best TPH removal rate 98.78% (IR) and 99.8% (CG).

Diversity of fungal endophytes determined in this study supports previous reports of hyperdiversity of this group of microorganisms in Ecuador. Our results suggest that bioremediation processes could potentially use hydrocarbon-degrading endophytic fungi isolated from native plant species growing in natural oil outcrops. These may also be used in support of phytoremediation strategies at later stages of remediation of contaminated sites given that the endophytes isolated from these plant species may present long-term adaptation mechanisms that can improve colonization of contaminated habitats, as well as uptake by plant species.

The authors wish to acknowledge financial support of this research by the Pontifical Catholic University of Ecuador. Plant and endophyte collections were carried out through the Ecuadorian Ministry of the Environment permit 001-14 IC-MC-DBN/MA. We thank Elisa Jara, School of Chemical Sciences at PUCE and the Center of Environmental and Chemical Services of the PUCE (CESAQ-PUCE) for assistance in the evaluation of hydrocarbon biodegradation. Special thanks to Carolina Portero for technical assistance and Javier Carvajal for valuable comments.

The authors declare no conflicts of interest regarding the publication of this paper.

Marín, F., Navarrete, H. and Narvaez-Trujillo, A. (2018) Total Petroleum Hydrocarbon Degradation by Endophytic Fungi from the Ecuadorian Amazon. Advances in Microbiology, 8, 1029-1053. https://doi.org/10.4236/aim.2018.812070