Optimal Parameters for in Vitro Development of the Fungus Hydrocarbonoclastic Penicillium sp

Marcia Eugenia Ojeda-Morales, Miguel Ángel Hernández-Rivera, José Gabriel Martínez-Vázquez, Yolanda Córdova-Bautista, Yuridia Evelin Hernández-Cardeño

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División Académica de Ingeniería y Arquitectura, Universidad Juárez Autónoma de Tabasco, Cunduacán, México.

DOI: 10.4236/aces.2013.34A1004                

Abstract

México has extensive areas that have been impacted by oil spills for several decades. Current bioremediation technologies mostly used microorganisms to decontaminate sites with hydrocarbons. This research evaluated the conditions for the optimal development of the strain of a hydrocarbonoclastic fungus, which was found in samples of soil contaminated with 4.0 × 10⁵ mg·kg^-1 of Total Petroleum Hydrocarbons (TPH). A completely randomized experimental design with a 3 × 3 × 4 factor arrangement was used: three levels of temperature (T₁ = 29℃, T₂ = 35℃ and T₃ = 40℃), three of pH (pH₁ = 3.5, pH₂ = 5.0 and pH₃ = 6.0) and four nutrients (N₁ = Urea, N₂ = Triple-17, N₃ = Nitrophoska-Blue and N₄ = Pure-Salts). Total fungi were isolated from the sampled soil and were sown in a combined carbon medium for hydrocarbonoclastic fungi and a strain was selected to be adapted to a liquid mineral medium. The selected strain was classified as Penicillium sp. Analyses of variance and mean tests were performed, using the SPSS-11.0 statistical software. The microorganisms showed the highest population growth in the treatment N₂pH₂T₁, which reached a value of 2.1 × 10⁶ CFU·mL^-1 in a biorreactor. To reach it, by bioaugmentation, the same development of Penicillium sp. in a conditioned soil would allow to implement a bioremediation strategy with great potential to retrieve soil contaminated with hydrocarbons both in Tabasco and in general in Mexico.

Keywords

Hydrocarbon Degraders; Optimal Growth; Penicillium sp.; Hydrocarbonoclastic

1. Introduction

The exploitation of energy resources in the state of Tabasco is of great importance for Mexico [1]. The total amount of crude oil extraction in 2009 in Mexico was 2.6 million of daily barrels. 77.5% of these barrels were obtained from the Mexican Gulf [2], and the other 22.5% were from wells in land. 77% of the petroleum extracted from the wells in land came from fields located in the state of Tabasco [3], which represented the 3.7% of the Gross National Product (GNP) [4]. However, oil industry accidents like rupture of pipelines and shipwrecks cause oil spills on land and marine ecosystems, producing pollution processes that affect the biophysical properties of soil and water and the biological components of these ecosystems. All this prevents the polluted natural resources from being fully exploited as well as health problems to the population living there [5,6]. Several studies including bioremediation techniques have been carried out in these sites, to stop the problems affecting the ecology and human health.   

Bioremediation uses the metabolic potential of microorganism to clean up an open polluted environment [7]. In the oil industry, it is becoming a widely used and cost effective technique to clean up hydrocarbons due to its simple use over large areas and its capacity to completely destroy the contaminant [8,9]. Within bioremediation, the bioaugmentation involves the inoculation of a strain or an enriched mixed microbial consortium in the soil [10-16]. It was found that indigenous microorganisms are especially efficient in the degradation of indigenous crude oil (produced in a determined region), but they are not that efficient in oils coming from other sites [17]. Native or indigenous microbes are present in small quantities and cannot prevent the contaminant from being spread; they do not have the capacity to degrade a particular contaminant or they can be in an inactive metabolic form in their habitats, which is why bioaugmentation is preferred over biostimulation [18]. Bioaugmentation offers an option to grow specific microbes in sufficient amounts to complete the biodegradation [15,16,19]. In this context, there exist a great number of heterotrophic microorganisms capable of using oil hydrocarbons as a source of carbon and energy, producing carbon dioxide, water, biomass and other less toxic products [20]. Among the group of fungi, the most extensive studies have focused on white rot fungi [21,22]. Filamentous fungi have some features that make them excellent agents of degradation. Once the microorganisms quickly ramify and the fungal hyphae penetrate the polluted soil, they reach the substratum and absorb it through the secretion of extracellular enzymes. They grow under stress conditions: under pH, lack of nutrients and low water activity [23]. Fungi have also demonstrated their ability to degrade in some cases, such as mineralize phenols, halogenated phenolic compounds, petroleum hydrocarbons, polycyclic aromatic hydrocarbons and polychlorinated biphenyls in large stress conditions [24,25].   

The Penicillium sp. fungi have been used in studies of crude oil biodegradation [16,23,25-28], and the mineralization of a variety of oil derivates, such as polycyclic aromatic hydrocarbons (PAHs), including pyrene, chrysene, and benzo [a] pyrene and polar metabolites [12,25, 29-31], as well as toxicity studies with hexadecane, phenanthrene and beta-naphthol, as biodegradation sometimes cannot be carried out because of the toxicity of the oil rather than its own persistence [9]. The metabolism of PAHs by strains of filamentous fungi is mediated by extracellular lignolytic enzymes or intracellular cytochrome P450 monooxygenases [32,33]. Both routes mainly produce quinones as oxidation products, which have higher solubility and reactivity than starting PAH [34-36]. In the case of the Penicillium fungi, the monooxygenase enzyme systems are responsible for degrading PAHs, where the first steps of oxidation are the formation of monophenols, diphenols, dihydrodiol and quinone [35]. In a second step, conjugates of O-methyl and sulfate can be formed; they are detoxification products soluble in water [37].  

It has been reported that fungi are better than bacteria in the degradation of hydrocarbons in both quantity and variety of constituents, indicating that the species of fungi most commonly found in soil and seawater contaminated with hydrocarbons are the genera Aspergillus and Penicillium [9,16,38,39]. The ability of Aspergillus and Penicillium sp. to tolerate these pollutants and grow in them, suggests they may be used as bioremediation agents and can be used in the restoration of the ecosystem when impacted by these pollutants, because they produce enzymes and acids that break and dismantle the long chains of hydrocarbons, the base structure common to oils, petroleum products and many other pollutants [29]. Despite the abundance of fungi, Penicillium in particular has received little attention in studies of biodegradation, with a relatively small number of pilot projects and large-scale studies using this organism that has a high potential in the field of bioremediation [25].

The objective of this study was to evaluate the optimal physicochemical and nutrient conditions that promote higher biomass growth of a hydrocarbonoclastic strain of the Penicillium sp., in order to have new strategies that allow the use of this strain in bioremediation by bioaugmentation in soil contaminated with crude oil or petroleum products, both in the state of Tabasco and throughout Mexico.

2. Material and Methods

The research was conducted in two stages: the first consisted of three phases where the process of isolation, purification, testing, characterization, and preservation of hydrocarbonoclastic microorganisms was developed.  

2.1. Stage 1. Assessment, Characterization and Adaptation of Hydrocarbonoclastic Fungi.

Samples from a Gleysol-mollic contaminated soil were collected in the facilities of an oil field located in the Huimanguillo municipality [40], state of Tabasco in Mexico, at an altitude of 20 meters above sea level [41]. Simple soil samples were taken, in accordance with the Mexican Official Standard 021 [42], for a total of 5000 m² (0.5 ha), and stored at 4.0˚C until use. Then, the Total Petroleum Hydrocarbons (TPH) present in the soil samples were determined by the method of extraction [43,44], in their heavy fraction (greater than C18) and then quantified by gravimetry [45]. Soil samples were subjected to sowing in Phase I.

2.1.1. Phase I

The process of isolation, purification and preservation of total fungi was based on their growth and reproduction in the Potato Dextrose Agar growth medium PDA (Baker). Prepared according to the manufacturer, it was poured in petri dishes and sterilized. A solution was prepared with 10.0 g of the sampled soil in 90 mL of sterile distilled water, from which two serial dilutions were prepared: 1 mL of this solution was added to 9 mL of sterile distilled water (10⁻¹), making and additional dilution, similar to the latter solution (10⁻²). Subsequently, 0.1 mL of the 10⁻¹ dilution was sown in each of 6 the petri dishes with PDA, spreading with a Drigalsky spatula. The same was done with the 10⁻² dilution and all of them were incubated at 28˚C for 8 days (d). The quantification of total fungi was performed by the plate counts method for viable cells by serial dilution [46]. Selection tests of fungi in the soil were based on radial growth in the growth medium. Among the 24 strains of fungi found, 5 with the highest development were chosen to be assessed as hydrocarbonoclastic in Phase II (H14, H18, H19, H20 and H24). These strains were preserved on PDA medium in inclined tube at 4˚C until use.

2.1.2. Phase II

Adaptation in Solid Medium Cellulose-Agar (SCA) for fungi to degrade total petroleum hydrocarbons. The SCA medium (Baker) was prepared according to Rivera-Cruz et al. [47], and poured into petri dishes. Culture media, Istmo crude oil with API index = 33.74 [48], in its heavy fraction (with molecules greater than C18) and the filter paper (squares with 1.5 cm on each side), were sterilized in wet heat during 20 minutes in autoclave at 121˚C and 127,486 kPa (1.3 kgf∙cm⁻²) [48]. A 1.5 × 1.5 cm filter paper impregnated with crude oil was placed on the SCA in the petri dishes under axenic conditions. On the other hand, the strains of fungi obtained from the previous phase were sown again. The mycelium of the preserved fungus colonies was touched with a handle and striated in the sterilized PDA medium prepared as indicated by the manufacturer. Then it was incubated at 35˚C for 72 h. For sowing in SAC, a slice of fungus with a reproductive structure of the sown strains was extracted with a puncher (0.9 cm in diameter) and was placed on filter paper impregnated with oil, then the Petri dish was covered [40]. Sowing was done separately and in triplicate in the SCA medium for each fungal strain and incubated at 29˚C for 6 d, with evaluation every 24 h. The three strains that showed more radial development, H14, H18 and H24 passed to the next phase.     

2.1.3. Phase III

Adaptation of fungi in liquid mineral medium Cellulose-Agar (LCA) for fungi to degrade Total Petroleum Hydrocarbons (TPH). Fungi selected in Phase II were sown in the LCA medium. This was prepared as suggested by Rivera-Cruz [40], with crude oil incorporated into the culture medium as carbon and energy source. Then each organism was sown separately, as proposed by Rivera-Cruz et al. [47]. The fungal growth was determined every 72 h for 6 d and H24 strain was selected because of its higher population growth. A micro-culture of H24 in PDA was performed, and with the keys of Barnett and Hunter [49]. The organism was identified as Penicillium sp. This fungus was preserved in PDA medium at 4˚C in inclined tube.     

2.2. Stage 2. Development of Experimental Design to Determine Optimal Growth Parameters

The second stage was conducted in two phases. In the first phase an in vitro bioassay was established where the fungus selected in the previous stage was subjected to four nutrient media, varying pH and temperature. In the second phase a test of fungal biomass production was done in a bioreactor (Kettler jar) with the parameters that stimulated an optimal development of the microorganism.     

2.2.1. Phase I

An in vitro bioassay was established, based on a completely randomized design in 3 × 3 × 4 factorial type, namely: three levels of temperature (T₁ = 29˚C, T₂ = 35˚C and T₃ = 40˚C), three pH levels (pH₁ = 3.5, pH₂ = 5.0 and pH₃ = 6.0) and four types of nutrients (N₁ = Urea, N₂ = Triple-17, N₃ = Nitrophoska-Blue and N₄ = PureSalts), under the statistical model of Equiation (1).  

(1)

Temperatures of 35˚C and 40˚C were established using two glass trays, each connected to an electric heater. Water temperature was kept constant using a submersible recirculation pump. To determine nutrients and pH parameters, 250 mL Erlenmeyer flasks were used, divided into three series of 12 treatments; they were connected to a compressor with filter membrane and valve controller for supplying sterile air.  

Preparation of culture media. Growth media used were: N₁ = Urea (Abbot), N₂ = Triple-17 NPK (Rancho Los Molinos), N₃ = Nitrophoska-Blue 12-12-17-6 NPKS (Compo) and N₄ = pure-Salts Na₂HPO₄, KH₂PO₄, NH₄Cl, MgSO₄∙7H₂O [50], prepared with equal proportions of C, based on the same amount of glucose (Merck), added to each medium. The fertilizer nutrients had proportional relations in the following NPK elements: proportion between Pure-Salts: Triple-17, N (approx. 1:1), P (approx. 4:1), K (approx. 2:1), respectively; proportion between Triple-17:Nitrophoska-Blue, P (2.8:1), N (2.8:1), K (1:1); proportion between Urea:Pure-Salts, N (3.4:1). Moreover, the mass load was the same for the three organic fertilizers. Media were prepared as follows: for each of the four media, three Erlenmeyer flasks with 500 mL of distilled water (Mercury Chemical) and 2.5 g of glucose as carbon source were prepared. The first medium contained 0.25 g of Urea, the second: 0.25 g of Triple-17, the third, 0.25 g Nitrophoska-Blue and fourth: 0.5325 g of Na₂HPO₄ (Fermont), 0.325 g of KH₂PO₄ (Fermont), 0.125 g of NH₄Cl (Golden Bell), 0.05 g of MgSO₄·7H₂O (JT Baker), making a total of 12 flasks. Then, each of these nutrient media was adjusted in pH to 3.5, 5.0 and 6.0 respectively, using 0.1 M of H₂SO₄ (JT Baker) and NaOH (JT Baker) solutions. To assess the nutrient media at each temperature, 150 mL of each nutrient medium with its pH adjusted were measured in triplicate and placed in a 250 mL flask, obtaining 36 experimental units (EU), according to experimental design. The EU were sterilized for 20 min at 121˚C and 103,421 kPa (15 lb_f∙plg⁻²). These EU were subsequently inoculated with Penicillium sp.

Preparation of inoculum: The Penicillium sp. Preserved (Phase III in Stage I) was re-isolated in Petri dishes on PDA medium prepared as indicated by the manufacturer, then it was sterilized; the mycelium of the fungus colonies preserved was touched with a handle and striated in the PDA medium. Then, it was incubated at 35˚C for 72 h. After that, the mycelium of the re-insulated fungus was touched with a handle and striated in the PDA medium in an inclined tube and incubated at 35˚C for 4 d. Then 5 mL of sterile distilled water was added in the inclined tube to allow separation of the spores. The above mixture was poured into an Erlenmeyer flask with 100 mL of water in axenic conditions. The initial spore concentration was determined in this volume by counting in a Neubauer chamber according to Pica et al. [51]. 2.5 mL of this inoculant medium was injected in each of the 36 EU under axenic conditions to homogenize and the initial amount of inoculum Colony Forming Unit (CFU) of Penicillium sp. was determined at time zero hours (t = 0 h). The evaluation of the Penicillium sp. growth in the EU was made every 2 d for 37 d by the method of counting viable cells by plating on surface [46]. Also, pH values and temperature were measured every 24 h during the time the experiment lasted.   

2.2.2. Phase II

Production of fungal biomass in a bioreactor (Kettler jar). The production of biomass Penicillium sp. was done in a 2000 mL bioreactor fitted with a mechanical stirrer, sterile air inlet, sample taking, and air outlet to release pressure. The optimal parameters for microbial growth from the previous phase: N₂ = Triple-17, pH₂ =5.0, T₁ = 29˚C were adjusted to prepare 1100 mL of nutrient medium that was sterilized in the bioreactor for 20 min at 121˚C and 103,421 kPa (15 lb_f/plg²), and 18.33 mL of inoculant prepared similarly to the previous phase was added. Then, it was shaken to homogenize and sampled to determine initial CFU number (t = 0 h). The evaluation of the development of Penicillium sp. was performed every 24 h for a period of 15 d. In both cases the quantification of CFU of the microorganism is determined similarly to the previous phase. Temperature and pH were measured during each evaluation of fungal growth.  

3. Results

The study was conducted on soil contaminated with Istmo crude oil (hydrocarbon chain greater than C18), with API = 33.74 [48], in order to find and isolate hydrocarbonoclastic microorganisms to be used in the production of fungal biomass. It was determined that the soil was contaminated with 4.0 × 10⁵ mg∙kg⁻¹ HTP (400,000 ppm). In the microbial isolation (Phase 1 of Stage 1), 24 strains of total fungi were found. Figure 1 shows the results for 14 strains of fungus. The 5 strains selected for further evaluation in Phase II were those with the highest radial development in the PDA culture medium.   

Sowing the 5 strains of the previous Phase in Solid medium Cellulose-Agar (SCA) for TPH degrading fungi (Phase II) resulted in a greater development for strains H14, H18 and H24. These 3 strains passed to phase III (Table 1).   

Table 2 (Phase III) shows the growth performance of the three selected fungi in Phase II in a mineral medium enriched with crude oil (LCA). Its speed behavior was observed over time (6 d). Likewise, it was verified that the crude oil surface tension cracked, completely chang-

Figure 1. Radial growth of the fungi on PDA medium.

Table 1. Radial growth of fungal strains in Solid medium Cellulose-Agar (SCA) to TPH degrading fungi.

Table 2. Growth of fungal strains as colony forming units (CFU) in Liquid mineral medium Cellulose-Agar (LCA), enriched with crude oil during the 6-d-long experiment.

ing its appearance. With these results, the three strains of fungi were identified as hydrocarbonoclastic. From these strains, the one named H24 and identified as Penicillium sp. by comparison with the keys of Barnett and Hunter [49] had the largest growth during the period of the experiment and was selected for evaluation in Stage 2.

In assessing the growth of Penicillium sp. in a flask (Stage 2, Phase I), an increase in the viscosity of the medium during the biomass growth was observed. With the data obtained the mean test was done.

Figure 2 shows the results of treatments at different temperatures, nutrients and pH. Figure 2(a) indicates that the growth of Penicillium sp. was the best at a temperature of 29˚C, but slightly less in the other two temperatures. Figure 2(b) indicates that the microorganism had a better development in the medium with Pure-Salts, but not very different from the media with NitrophoskaBlue and Triple-17. The lowest growth was in the medium with Urea. Figure 2(c) shows that the population growth of Penicillium sp. was higher at pH 3.5 but not so different to that achieved in the other two pH values. 

Conflicts of Interest

The authors declare no conflicts of interest.

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