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Ciencia e investigación agraria

On-line version ISSN 0718-1620

Cienc. Inv. Agr. vol.43 no.1 Santiago Apr. 2016

http://dx.doi.org/10.4067/S0718-16202016000100008 

 

ENVIRONMENT AND ECOLOGY/RESEARCH PAPER

 

Tolerance of native Magellan fungi in peat to anthracene and n-dodecane for potential use in bioremediation

 

Leonardo A. Rodríguez-Córdova1, César A. Sáez-Navarrete1, Vartan Ishanoglu2, Leandro Herrera3, and Rosanna Ginocchio4

1Departamento de Ingeniería Química y Bioprocesos, Escuela de Ingeniería, Pontificia Universidad Católica de Chile. Ave. Vicuña Mackenna 4860, Macul, Santiago, Chile.

2Dirección de Responsabilidad Social, Escuela de Ingeniería, Pontificia Universidad Católica de Chile. Ave. Vicuña Mackenna 4860, Macul, Santiago, Chile.

3Departamento de Ingeniería Química y Biotecnología, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Beauchef 851, Santiago, Chile

4Departamento de Ecosistemas y Medio Ambiente, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad Católica de Chile, Ave. Vicuña Mackenna 4860, Macul, Santiago, Chile.

Corresponding author: larodrig@uc.cl


Abstract

Tolerance of Magellan fungi in peat to anthracene and n-dodecane for potential use in bioremediation. Cien. Inv. Agr. 43(1):85-93. The tolerances of filamentous Magellan fungi in peat were analyzed in the presence of structurally different petroleum hydrocarbons to assess their abilities for bioremediation in contaminated soils. Fungi on PDA (potato dextrose agar) and fresh peat plates were cultured and purified. The morphologically identified species were grown in a hydrocarbon adapted-fungi (HAF) medium. Increasing concentrations of anthracene, n-dodecane and furfural were applied to observe tolerances based on the radial growth kinetics of hyphae. The results showed that the radial growths of hyphae in anthracene reached average speeds of 10.95 ± 1.21, 11.03 ± 3.14 and 10.96 ± 4.61 mm h-1 in 0.1, 1 and 2 g L"1 solutions, respectively. The average growth rates in n-dodecane were 10.52 ± 3.33, 14.67 ± 1.88 and 10.86 ± 3.50 mm h-1 in 10, 20 and 40 g L-1 solutions, respectively. The growth rate in furfural reached 3.95 ± 1.07 mm/h in a 5 g L-1 concentration. The results suggest that the identified filamentous fungi are tolerant to anthracene and n-dodecane, which are the primary components of petroleum fractions. Furfural, a recognized antifungal, limited the growth. The results also indicate hydrocarbon degradation, suggesting that Magellanic peat can be used as a potential inoculum in bioremediation treatment processes associated with petroleum-contaminated soils. The observed filamentous fungi belong to the Penicillium genus based on visual identification and 18S rRNA.

Key words: Anthracene, contaminated sites, filamentous fungi, furfural, Magellan peat, n-dodecane


Resumen

Tolerancia a antraceno y n-dodecano de hongos nativos de turba Magallánica para su potencial uso en biorremediación. Cien. Inv. Agr. 43(1):85-93. Se estudia la tolerancia de hongos filamentosos de turba magallánica en presencia de hidrocarburos derivados del petróleo estructuralmente diferentes, con miras a ser empleados como agentes de biorremediación de suelos contaminados. Se cultivaron y purificaron hongos en placas con PDA (Agar de Papa y Dextrosa) y turba fresca. Las especies morfológicamente identificadas se cultivaron en medio HAF (Hidrocarbon Adapted-Fungi) con concentraciones crecientes de antraceno, n-dodecano y furfural para observar su tolerancia a través de las cinéticas de crecimiento radial de hifas. Los resultados muestran que el crecimiento radial de las hifas en antraceno alcanzó una velocidad media de 10.95 ± 1.21, 11.03 ± 3.14, y 10.96 ± 4.61 mm h4 para concentraciones de 0.1, 1 y 2 g L4; para n-dodecano las velocidades medias alcanzaron los 10.52 ± 3.33, 14.67 ± 1.88 y 10.86 ± 3.50 mm h4 para 10, 20 y 40 g L4. Ensayos con furfural alcanzaron 3.95 ± 1.07 mm h4 a 5 g L4. Los resultados muestran que el hongo filamentoso identificado es tolerante a antraceno y n-dodecano, constituyentes característicos de fracciones del petróleo. Por su parte furfural, reconocido antifúngico, limita el crecimiento. Los resultados también muestran degradación de los compuestos señalados posicionando a la turba Magallánica como potencial contribuyente de inóculos para procesos de biorremediación de suelos contaminados con hidrocarburos del petróleo. Se identificó el género Penicillium como el hongo filamentoso desarrollado mediante identificación visual y 18S RNA.

Palabras clave: Antraceno, furfural, hongos filamentosos, n-dodecano, sitios contaminados, turba Magallánica.


 

Introduction

The intensive use of petroleum fuels has been the basis for industrial development since the nine-teenth century. Projections for 2035 indícate that fossil fuels may provide 80% of the world energy needs (OPEC Secretariat, 2013). However, massive crude oil extraction processes, the production of hydrocarbon compounds derived from refining operations, oil storage and fuel transportation have resulted in spills and emissions that introduce xenobiotics, toxic and hazardous chemicals into the biosphere. Many of these toxins are highly persistent in aquatic and terrestrial environments (Blinova et al, 2012). To reduce these impacts, countries have developed increasingly strict envi-ronmental and operational regulations as well as more efficient and effective control strategies for spills and emissions (Deuel et al, 2005). These strategies have improved production processes and existing technologies. Additionally, several technologies have been employed to remediate petroleum-contaminated soils. Among these technologies, those based on the use of native microorganisms grown at the impacted site have become particularly popular (Godoy-Faúndez et al, 2008; Sáez-Navarrete et al, 2008). The idea behind this method is to prevent the introduction of new competitor species, which may jeopardize the biological balance of the soil; therefore, the method uses native species that are adapted to the environmental conditions of the site and, in many cases, to the contaminants (El Fantroussi and Agathos, 2005).

Sources for obtaining these microorganisms are diverse, but organic substrates are preferred due to their ecological diversity (Godoy-Faúndez et al. 2008; Sáez-Navarrete et al, 2008). In such substrates, complex microecosystems formed by bacteria, fungi, yeast, actinomycetes and microalgae can be identified. These constituents can potentially transform contaminants into less hazardous chemicals or even to carbon dioxide (Beskoski et al, 2012). Peatlands are included among these microecosystems. Peat is a spongy, carbon-rich organic matter that is produced by the decomposition and fossilization of plant material (Sáez-Navarrete et al, 2008). Its characteristics vary spatially and depend on the local plant spe-cies, representing a rich biological stratification (Bragina et al, 2014).

In Chile, peatlands are concentrated in the south of the country, encompassing a total area of 10,470 km2. The most common peatland type is found in Chilean Patagonia, covering a total of 90,000 ha (MMA, 2015). The dominant species is sphagnum moss, which has been previously used as a bulking material and nutrient source for contaminant degraders in remediation studies of petroleum-contaminated soils (Sáez-Navarrete et al, 2008). Microorganism studies near Chilean Patagonia and Antarctica have noted that Magel-lanic peat can potentially be used to treat petroleum hydrocarbon contamination (Vazquez et al, 2013) in cold regions and also in situ. Some species of filamentous fungi present in Magellanic peat may be able to not only tolerate complex carbon-based compounds but also mineralize and biodegrade them, as has been described for other substrates (Antízar-Ladislao et al, 2004).

The purpose of this study is to identify native Magellanic fungi in peat that are tolerant to an-thracene, n-dodecane and furfural and possess potential for degradation and bioremediation in contaminated soils.

Materials and methods

Fungal strains and hydrocarbon selection

Fresh peat was extracted from the Magallanes Region and the subantarctic territory (Punta Arenas, 53°09’00" S, 70°55’00" W). The samples were obtained at 20 to 30 cm depth using sterile and airtight plastic bags. The samples were trans-ported in cold medium to later perform growth testing in the presence of the contaminants of interest. Based on the methodology described by Hughes et al. (2007), two peat samples were collected (0.35 g wet weight each) and placed in dextrose saturated water (10 mL) in Petri dishes in triplicate (6 plates). Plates were incubated for 15 days at 28 ± 2 °C, allowing enough time for the growth of filamentous fungi. Incubation was performed in the dark at a relative humidity of 17 ± 2%.

The developed hyphae samples were placed in Petri plates with potato dextrose agar (PDA) and antibiotics. The plates were incubated at 28 ± 2 °C for 20 days. Preliminary identification of the filamentous fungi was performed using a Motic® BA310 optical microscope based on the descrip-tions of their morphological characteristics.

Anthracene, furfural and n-dodecane were chosen as the selected contaminants. N-dodecane was used as a representative of biodegradable alkanes. Furfural was used as a representative of heterocyclic aromatic hydrocarbons. Anthracene was selected as a representative of low molecular weight PAHs due to its hazardous toxic characteristics (United States Environmental Protection Agency, 2006). Sigma-Aldrich brand reagents were used in the analysis, including furfural CAS No. 98-01-01 (99%), anthracene CAS No. 120-12-7 (99%) and n-dodecane CAS No. 112-40-3 (99%).

Tolerance tests

To establish the tolerance of the fungal species, hydrocarbon-adapted fungi (HAF) (Elshafie et al., 2007) were used in sterile Petri dishes. The basal components of the HAF medium consist of 250 mg L-1 of KCl, 1 g L-1 of NaH2PO4, 1 g L-1 of NH4NO3 and 15 g L-1 of agar. A contaminant was then added to each medium. HAF plates without contaminants were used as controls.

All selected hydrocarbons were used at concentra-tions of 0.1, 1 and 10 g L-1, respectively. Experiments were performed in triplicate. A HAF medium with agar as the sole carbon source was used as a control. Initially, Petri dishes contained the HAF media plus contaminants at the concentrations indicated above. The plates were filled to half their volumetric capacities. Subsequently, 0.35 g of peat was placed on each plate. The plates were developed at 28 ± 2° C for 15 days in the dark to allow fungal growth. A subculture with observable hyphae was then created. To do so, the same HAF media and selected contaminants in the concentrations indicated above were used as purifying mechanisms of tolerant strains. In this subculture, radial growth and morphological characteristics were observed under a microscope.

Tolerant fungal strains

After tolerance testing, hyphae were removed to carry out genetic characterization of those fungal species developed in the experiment, using 18S rRNA (Nishida and Sugiyama, 1993).

Statistical analyses

The differences in the radial growth in each condition studied (treatment) were evaluated by two-way ANOVA using time and treatment as factors (Akbari and Ghoshal, 2014). A statistical analysis was performed using Minitab software with a = 0.05. Normality, independence and homoscedasticity of the data were also verified.

Results and discussion

Fungi observations

After the initial incubation, hyphal growth in peat (Figure 1a) was observed. These hyphae quickly colonized the plates where they were inoculated (Figure 1b), covering 100% of the available surface in two days (Figure 1c).

Samples were extracted from the fully colonized Petri dishes to be identified using conventional optical microscopy. Morphological characterization showed a single type of strain with characteristics similar to those of Penicillium. Conidiophores (main stem), metulae (outer branch) and spores were observed, and phialides formed at the end of the conidiophores and metulae (sporulation area shaped like a brush). These observations are consistent with Penicillium. Furthermore, a teal color, which is characteristic of this genus, was described. No other strains were observed under the microscope.

After analyzing the characteristics of this filamen-tous fungus, they were compared to microscopic images of filamentous fungi of Penicillium avail-able in the literature. This comparison confirmed the initial hypothesis regarding the presence of this species (Figure 2).

While Penicillium is common in temperate envi-ronments, it has also adapted to very cold areas. Indeed, samples obtained for this study were obtained from peatlands located in Patagonia and in the subantarctic territory of Chile. This fungal species has also been identified in Magellan soils contaminated with petroleum and petroleum prod-ucts (Balaji et al., 2014), in which its degradative potential has been described. This species produces cellulases, which are nonspecific enzymes capable of degrading complex compounds such as cellulose via osmotrophy (Harms et al., 2011).

Strain tolerance

Controlled fungal cultures and treatments are shown in Figure 3. The availability of the sole carbon source, agar-agar, promoted radial growth at an average of 6.89 ± 2.09 mm h-1 (control). The lag phase observed is 15 h.

The experiments using HAF, anthracene, dodec-ane and furfural exhibited growth rates within normal ranges. The radial growth for 5 g L-1 of furfural displayed an average speed of 3.95 ± 1.07 mm h'1 (Figure 3). Furfural is a known commercial antifungal and antibacterial organic compound that has been associated with a strong antibiotic action and a lesser antifungal action at a concentration of 1 mM (0.096 g L-1) (Kurita et al, 1981). These results suggest that a furfural concentration of 10 g L-1 was necessary to inhibit fungi growth.


Figure 1. a. Fungal growth in peat. b. Fungi isolated from peat in a PDA medium (Potato Dextrose Agar) with antibiotics. c. Complete fungal colonization of the Petri dish.


Figure 2. a. Microscopic image of Penicillium sp. SA29 (Ohashi et al., 2008). b. Microscopic image (40x) of filamentous fungi obtained from Magellan peat.

 

Growth kinetics observed for dodecane (n-dodecane) were significantly higher than those of the control (P=0.009), reaching average growth speeds of 10.52 ± 3.33, 14.67 ± 1.88 and 10.86 ± 3.50 mm h-1 for concentrations of 10, 20 and 40 g L-1, respectively. The stereochemical structure of n-dodecane is susceptible to single bond attacks by nonspecific enzymatic activity associated with the fungi, which is able to degrade complex organic compounds such as cellulose, hemicellulose and lignin (Harms et al., 2011). This molecular structure makes it more metabolically assimilable than the polysaccharides present in agar (Figure 3). No significant differences were observed among the three n-dodecane concentrations tested (P=0.186).

The growth kinetics observed for anthracene were significantly higher than those of the control (P=0.003), reaching average growth speeds of 10.95 ± 1.21, 11.03 ± 3.14 and 10.96 ± 4.61 mm h-1 for concentrations of 0.1, 1 and 2 g L-1, respectively (Figure 3). Although significant differences among the tested anthracene concentrations (P=0.227) were not observed, the observed kinetics were found to be greater than those of the control.

Neither the growth in plates supplemented with the linear alkane nor in those supplemented with anthracene showed significant differences at the concentrations used (P=0.330). In fact, significant differences were not observed with increases of either organic compound, suggesting that the maximum fungal growth rates were reached for the carbón sources used (n-dodecane and furfural). Toxic and inhibitory effects were not observed at the concentrations tested.



Figure 3. The average radial growth of Penicillium on the control and furfural plates (5 and 10 g L"1) compared to: (1) Dodecane plates (10, 20 and 40 g L"1) and (2) Anthracene plates (0.1, 1 and 2 g L"1). Standard deviations of each group of three repetitions are included.

 

The fungal species tested can tolerate high concentrations of n-dodecane and anthracene, which are both known to be highly toxic. Based on the observed kinetics, it is likely that both hydrocar-bons were biodegraded by the fungal species. However, based on the experimental design and focus, the fraction of each hydrocarbon species that was degraded cannot be precisely determined with respect to the carbon source in the HAF medium, which was used as a control.

Identification of tolerant fungal strains

Genetic characterization based on 18S r-RNA identified the fungi as Penicillium. Three species of this genus were found. The fungus strains belong to the species Penicillium purpurogenum, Penicillium rugulosum and Penicillium variabile (Figure 4). Penicillium purpurogenum has been studied because of its metabolites and potential industrial applications, as its enzymatic process can be used in the production of biodiesel (Gusa-kov and Sinitsyn, 2012). Penicillium rugulosum has been found in arctic soils, and its enzymatic metabolism involves pectinases and cellulases


Figure 4. Phylogeny of Penicillium identified in Magellan peat.

(Gawas-Sakhalkar and Singh, 2011). Penicillium variabile has been studied in the context of phytohormone production (Isa and Don, 2014) and due to its gallotannin metabolism (tannase) (Sharma et al, 2008).

The genus Penicillium is known for its ability to degrade lignocellulosic materials (Leitão, 2009). Additionally, it is not surprising to find Penicillium in Magellan peat in a subantarctic area, as extremophile species of this fungus have been previously identified in cold regions (Gunde-Cimerman et al, 2003). The presence of this type of microorganism in a region that has been extensively contaminated by oil extraction since the early twentieth century is beneficiai for feasibility programs targeting bioremediation at these contaminated sites.

We conclude that the fungai species identified and tested can tolerate high concentrations of n-dodecane and anthracene, which are known toxins. Additionally, in terms of the observed kinetics, it is likely that both hydrocarbons are biodegraded by the fungal species studied. However, this experimental design focused on determining tolerance. Therefore, the degraded fractions of each hydrocarbon species cannot be precisely determined with respect to the carbon source contained in the HAF medium (control). The identification of these strains as Penicillium species demonstrates the degrada-tion potentials of the compounds tested, which should be further studied using Magellan peat as an inoculum.

Acknowledgments

This research was partly supported by the National Commission for Scientific and Technological Research (CONICYT) of the Chilean Government via a doctoral scholarship awarded to Leonardo Rodríguez-Córdova (No. 21,090,472). Thank you for your important contribution.

We thank the Laboratory of Biochemistry, Department of Molecular Genetics and Microbiology, Pontificia Universidad Católica de Chile, led by Professor Patricio Arce, who made an exceptional contribution to the genetic characterization tests.

We also thank Professor Loreto Muñoz, Micros-copy Laboratory, Department of Chemistry and Bioprocess Engineering, Pontificia Universidad Católica de Chile, for her kind and experienced assistance regarding the optical microscopy im-ages presented in this article.

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Received August 5, 2015.
Accepted January 13, 2016.

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