Received: 17 February 2018
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Revised: 15 April 2018
DOI: 10.1002/jobm.201800080
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Accepted: 22 April 2018
RESEARCH PAPER
Biodegradation of asphaltene and petroleum compounds by a highly potent Daedaleopsis sp. Elaheh Pourfakhraei1 Mahboobeh Nazari2
| Jalil Badraghi1
| Fatemeh Mamashli1 |
| Ali Akbar Saboury3
1 Research
Institute of Applied Sciences, Academic Center of Education, Culture and Research (ACECR), Shahid Beheshti University, Tehran, Iran 2 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 3 Institute
of Biochemistry and Biophysics, University of Tehran, Tehran, Iran Correspondence Jalil Badraghi, PhD, Department of Physics, Research Institute of Applied Sciences, ACECR, Shahid Beheshti University, Tehran, Iran. Email:
[email protected]
Petroleum, as the major energy source, is indispensable from our lives. Presence of compounds resistant to degradation can pose risks for human health and environment. Basidiomycetes have been considered as powerful candidates in biodegradation of petroleum compounds via secreting ligninolytic enzymes. In this study a wooddecaying fungus was isolated by significant degradation ability that was identified as Daedaleopsis sp. by morphological and molecular identification methods. According to GC/MS studies, incubation of heavy crude oil with Daedaleopsis sp. resulted in increased amounts of C24 compounds. Degradation of asphaltene, anthracene, and dibenzofuran by the identified fungal strain was determined to evaluate its potential in biodegradation. After 14 days of incubation, Daedaleopsis sp. could degrade 93.7% and 91.2% of anthracene and dibenzofuran, respectively, in pH 5 and 40 °C in optimized medium, as revealed by GC/FID. Notably, analysis of saturates, aromatics, resins, and asphaltenes showed a reduction of 88.7% and 38% in asphaletene and aromatic fractions. Laccase, lignin peroxidase, and manganese peroxidase activities were enhanced from 51.3, 145.2, 214.5 U ml−1 in the absence to 121.5, 231.4, and 352.5 U ml−1 in the presence of heavy crude oil, respectively. This is the first report that Daedaleopsis sp. can degrade asphaltene and dibenzofuran. Moreover, compared to the reported results of asphaltene biodegradation, this strain was the most successful. Thus, Daedaleopsis sp. could be a promising candidate for biotransformation of heavy crude oil and biodegradation of recalcitrant toxic compounds. KEYWORDS asphaltene, biodegradation, Daedaleopsis sp, heavy crude oil, ligninolytic enzymes
Abbreviations: ABTS, 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); AMDIS, automatic mass spectral deconvolution and identification system; ANT, anthracene; DBF, dibenzofuran; GC/FID, gas chromatography with Flame ionization detector; GC/MS, gas chromatography with mass spectrometry; HC, heavy crude oil; ITS, internal transcribed spacer; NIST, National Institute of Standards and Technology; PAHs, polycyclic aromatic hydrocarbons; PCR, polymerase chain reaction; SARA, saturated, aromatic, resins, and asphaltenes; TPH, total petroleum hydrocarbons. J Basic Microbiol. 2018;1–14.
1 | INTRODUCTION Petroleum is the major energy resource used by industry and in our daily lives [1]. It consists of saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes, making the acronym SARA. Many of petroleum compounds are highly toxic for living organisms [2]. Asphaltene fractions contain sulfur, oxygen, nitrogen, and small amounts of metal
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elements, such as Fe, Ni, and V [3,4]. Aromatic rings, acyclic, and heterocyclic structures are usually bonded to aliphatic hydrocarbons in asphaltene molecules. Due to formation of aggregates of several molecules, asphaletene's molecular weights can be increased and this is the most important problem in heavy crude oil (HCO) refinery [3,4]. Additionally, Asphaletene's extremely complex molecular structure causes them to be resistant to degradation in the environment [4]. In fact, petroleum production, transportation, storing, or using cause releasing of the hydrocarbons into the environment, accidentally, or operationally [1]. In addition to asphaltene, petroleum includes polycyclic aromatic hydrocarbons (PAHs) [5]. PAHs, the main cause of concern as anthropogenic environmental pollutants, pose risks to human health due to their various carcinogenic, mutagenic, and genotoxic activities [6]. Wastewater effluents from petroleum refineries and leakage from storage tanks and pipe lines could cause entrance of PAHs into the environment. Besides, owing to their low water solubility, PAHs are persistent in the environment [7]. Anthracene (ANT) and dibenzofuran (DBF), tricyclic, and two cyclic aromatic compounds, respectively, are two of the most persistent environmental toxic contaminants [8,9]. Biodegradation of petroleum hydrocarbons by bacteria, fungi, and yeasts is regarded as the main mechanism of eliminating hydrocarbon pollutants from the environment [10–12]. Very few microorganisms have been reported to biodegrade asphaltenes as the most recalcitrant crude oil fraction [13]. The reports included biodegradation or mineralization of asphaletes by mixed bacteria [13,14] and recently by Neosartorya fischeri [15], and Pestalotiopsis sp. [16,17]. Fungi could play an important role in biodegradation of petroleum compounds because fungi can form extensive mycelial networks and grow independently of pollutants as a growth substrate. Moreover, they have catabolic enzymes with low specificity, and the biochemical and ecological capacity [18]. In fact, ascomycetes and particularly basidiomycetes are prominent options in biodegradation of heavy molecules through producing ligninolytic enzymes. White rot fungi are a group of basidiomycetes that can degrade lignin and lignin-like substances [18–20]. Lignin is a recalcitrant and irregular polymer [21,22], similar in shape to asphaltene molecules found in HCO. Lignin is a phenolic heteropolymer but asphaltene contains aromatic, aliphatic, and heterocyclic compounds [23]. The lignin degrading system includes nonspecific oxidative exoenzymes, such as laccases and high redox potential ligninolytic peroxidases including lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) [24]. Having broad substrate specificity caused them to be useful in various biotechnological applications [25]. As a matter of fact, ligninolytic enzymes have been exploited in degradation of HCO and toxic compounds. Oxidation of a highly diverse
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array of organic and inorganic compounds such as PAHs and chlorinated aromatic compounds, pesticides, dyes, cyanides, and azides are catalyzed by these enzymes [23,26]. Several genera of basidiomycetes such as Phanerochaete, Pleurotus, Bjerkandera, and Daedalopsis have been shown to have potential biodegradative activities [19,27]. In an attempt to identify more potent fungal strains effective in HCO upgrading and biodegradation, our group conducted the current research. The purpose of this study is to introduce a fungal strain possessing strong biodegradative abilities to convert heavy molecules in HCO and toxic compounds.
2 | MATERIALS AND METHODS 2.1 | Chemicals HCO was a gift from Research Institute of Petroleum Industry with API = 18.5. NaCl, K2HPO4, KH2PO4, Tween 80, acetic acid, Tris base, Sabouraud dextrose agar (SDA), Malt Extract Agar (MEA), MnSO4, sodium acetate, sodium tartrate, hydrogen peroxide, glucose, sucrose, starch, sodium nitrate, peptone, yeast extract, (NH4)2SO4, KH2PO4, MgSO4, CaCl2, and KCl were purchased from Merck Company. Lactophenol cotton blue, 3, 4-dimethoxybenzyl alcohol (veratryl alcohol), catalase, 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ANT, and DBF were obtained from Sigma– Aldrich Company. Otherwise, it was mentioned in the text.
2.2 | Collection of fungal strains Fruiting bodies of fifteen wood-decaying fungal strains were collected from Chehel Chai and Nilkuh forests, Minudasht, Golestan province, Iran and were called EF1 to EF15. Each fungal strain was washed with serum physiology (0.9% NaCl), PBS (pH 7.4), and Tween 80 (0.1%) prior to inoculation to the culture media in order to remove the dust. Then they were cut into small pieces and placed on Petri dishes containing SDA. The plates were incubated in 30 °C for 72 h. The plates were maintained at 4 °C and were sub-cultured every two months. For long time storage, fungal strains were maintained at −70 °C.
2.3 | Culturing fungal strains in the presence of heavy crude oil Three agar plugs with diameter of 7 mm of every fungal strain were cultured in flasks containing 70 ml Czapek minimal medium without original carbon source (3.0 g L−1 NaNO3; 1.0 g L−1 K2HPO4; 0.5 g L−1 MgSO4•7H2O; 0.5 g L−1 KCl; and 0.01 g L−1 FeSO4•7H2O), and 1 ml HCO as carbon source [13]. The flasks were incubated in 30 °C and 120 rpm for 20 days. Uninoculated sterile broth flasks were used as control to assess HCO's biodegradation by fungal strains.
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2.4 | Measuring surface tension The surface tension of various cultures was measured via a Platinum Du Nouy ring method employing a digital tensiometer (Sigma 703D). The supernatant was used to evaluate surface tension following isolation of fungal mycelia by centrifugation at 10,000 rpm [28]. The strain presenting the lowest surface tension was selected for biodegradation studies.
2.5 | Chromatographic analyses Total petroleum hydrocarbons (TPH) contained in the culture media were extracted with n-hexane prior to analysis with gas chromatography with flame ionization detector (GC/FID; Agilent 7890N) equipped with an HP-5 fused silica column (30 × 0.25 m). The injector and detector were maintained at 300 °C, and the oven temperature was programmed to rise from 80 °C to 280 °C in 15 °C min−1 increments and to hold at 280 °C for 5 min. The flow rate was 1.5 ml min−1 and the injection volume was 1 µL. Furthermore, GC/MS analysis was performed to quantitatively determine the compounds formed following incubation of HCO with the selected fungal strain. The TPH fractions were extracted using chloroform and then analyzed by Agilent 6890 GC/MS equipped with HP-5 ms capillary column (30 m × 0.25 mm × 0.5 µm; Agilent Technologies) and an Agilent 5973 mass selective detector (70 eV). Helium was used as a carrier gas with a flow rate of 1 ml min−1. The temperatures of injector and detector were both 300 °C. Injection volume of 1 µL was applied. Different programs of oven temperature were examined, of which the following resulted in improved resolution: initial temperature of 70 °C for 1 min and then raised to 300 °C at a rate of 5 °C min−1 which was held for 20 min. The total area under the resulting chromatograms were noted and the chromatograms were analyzed by an automatic mass spectral deconvolution and identification system (AMDIS) to identify the petroleum components and compared against the National Institute of Standards and Technology (NIST) library in the GC/MS.
2.6 | Identification of fungal strain In order to identify the selected strain, the fruiting bodies were analyzed macroscopically and microscopically. The observations were recorded and compared with the scientific published data [29]. The selected strain was cultured in SDA, MEA, and Czapek at 30 °C for 14 days. The color, growth rate, general aspects, and hyphae structure were examined by stereomicroscope (Olympus optical SZH-ILLB; Japan). Furthermore, the strain was cultured on SDA, stained with lactophenol cotton blue, and observed microscopically (OPTIKA N-400FL; Italy) [30].
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Fungal DNA extraction was performed using i-genomic BYF kit (Intron, Korea). Molecular identification was performed based on analysis of internal transcribed spacer (ITS) region which include 5.8S, 18S, and 28S rRNA using universal primers [31]. The forward primer ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and the reverse primer ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (Metabion, Germany) were used to amplify ITS region through polymerase chain reaction (PCR) [32] performed by a CG1-960 Corbett Research Thermocycler. The 25 µL reaction mixture contained 12.5 µL master mix (Ampliqon, Denmark), 1 µL of each primer, 3 µL of extracted DNA, and 7.5 µL deionized water. After performing gradient PCR, 55 °C was chosen as the best annealing temperature. The reaction involved initial denaturation at 94 °C for 5 min, followed by 35 cycles in series of denaturation at 94 °C for 45 s, primer annealing at 55 °C for 45 s, and extension at 72 °C for 1 min, with a final extension step at 72 °C for 10 min. PCR products were electrophoresed on a 1% agarose (Life Technologies, USA) with TAE buffer, and analyzed after staining with DNA Green Viewer (Parstous, Iran). The PCR products were sequenced by Macrogen Company (South Korea). To identify the isolated genes, the sequences were analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). The neighbor-joining (NJ) method was used for phylogenetic analysis and was conducted using the MEGA version 6.06 software with the Tamura-Nei model [33]. ITS-rDNA of EF5 was submitted to GenBank and the accession no. is KY748235.
2.7 | Biodegradation of anthracene and dibenzofuran The ability of the selected strain was further evaluated in biodegradation of ANT and DBF as important PAHs. The selected strain was cultured in Czapek minimal medium without original carbon source (Please see “Culturing fungal strains in the presence of heavy crude oil” section) and 1000 ppm ANT or DBF as the sole carbon source [13]. Following incubation for 14 days, ANT or DBF were extracted using chloroform and was analyzed through GC/FID. The degradation percent was obtained according to the following equation: Degradation % ¼
Peak area of treated ANT or DBF 100 Peak area of the corresponding control
Afterwards, the culture conditions in terms of carbon and nitrogen sources and pH and temperature were optimized in further experiments.
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2.8 | The effect of pH and temperature on biodegradation of ANT and DBF Biodegradation ability of the selected fungal strain on ANT and DBF was evaluated in various temperature (20, 30, 40, 50, and 60 °C) and pH (3, 5, 7, and 9) conditions in order to determine the optimized temperature and pH. Briefly, the fungal strain was cultured in Czapek minimal medium in the absence of the original carbon source and instead presence of 1000 ppm ANT or DBF as the sole carbon source. Following 14 days of incubation, the PAH content of all the flasks were extracted using chloroform as the solvent. Afterwards, the extracts were analyzed by GC/FID.
2.8.1 | The effect of carbon and nitrogen sources on biodegradation of ANT and DBF Biodegradation ability was studied using three carbon sources such as glucose, sucrose, and starch and three nitrogen sources including sodium nitrate, peptone, and yeast extract in the concentration range of 5–20 g L−1. Other materials were also added to all the flasks including 2 g L−1 (NH4)2SO4, 2 g L−1 KH2PO4, 0.6 g L−1 MgSO4, 0.2 g L−1 CaCl2, 0.05 g L−1 KCl, and 1000 ppm of ANT or DBF . All the culture media were autoclaved at 121 °C for 20 min. Afterwards, the flaks were inoculated with the selected fungal strain followed by incubation for 14 days in 40 °C, pH 5, and 120 rpm. Finally, the PAH content of all the flasks were extracted by chloroform followed by GC/FID analysis and calculation of the percent of biodegradation.
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supernatant were included in all the tests. The enzymatic activities were measured in 30 °C after 3 min [37,38]. The data were represented as means of three independent experiments. One unit of enzymatic activity was defined as the amount of enzyme that converts one micro mole of substrate per minute to product.
2.10 | SARA analysis Saturates, Aromatics, Resins, and Asphaltenes (SARA) methodology was applied to analyze the various components of crude oil. At the first step, the extracted TPH was dissolved in n-heptane which resulted in the precipitation of the asphaltene fraction. The soluble fraction containing resins, aromatics, and saturates was separated using a silica gel column. Saturates and aromatics were eluted with n-hexane, and toluene, respectively. Asphaltene was eluted with 80:20 v/v dichloromethane/methanol mixtures. Following collection, the mass of each fraction was determined gravimetrically. Each sample was analyzed three times [39].
2.11 | Statistical analysis Statistical calculations were carried out using SPSS version 24 statistical software (SPSS Inc., USA). One-way ANOVA and Duncan's multiple range tests were used for mean comparisons. Furthermore, a p-value of C24) has reduced. Furthermore, C29, C33, and C34 have totally been degraded while C25-28 and C30-32 have been partially degraded. Interestingly, increased ratio of C9-24 indicates emergence of shorter chain hydrocarbons as a result of biodegradation of HCO by EF5. Besides, C9, C11, C10, and C15 presented the highest ratio in order. In general, incubation of HCO with EF5 has resulted in increased shorter chian hydrocarbons.
GC/FID chromatograms of TPH obtained from liquid cultures: temperature 30 °C and agitation speed of 120 rpm. (a) Control in the absence of EF5; (b) sample in the presence of EF5
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FIGURE 3
GC/MS chromatograms of TPH obtained from liquid cultures: temperature 30 °C and agitation speed of 120 rpm. (a) Control in the absence of EF5; (b) sample in the presence of EF5
3.3 | Identification of fungal strain The selected strain EF5 was identified through both morphological and molecular techniques. Fruit body was fan-shaped and leathery with a thickness of about 2 cm. Moreover, the upper surface of the fruit body was smooth, dry, and full of cylindrical pores. EF5 presented a growth rate
according to the following order: Czapek > MEA > SDA. In the case of SDA, the young colony was white and it turned light orange after some days. Furthermore, the colony was cottony, smooth, and formed a radius of 2.2 cm around the inoculum. In the case of MEA, the colony was convex and wavy and it turned darker after some days. In the case of Czapek, the colony was larger (a radius of 2.8 cm) and also the hyphae presented a
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TABLE 1 Degradation of hydrocarbons with different chain length in heavy crude oil by EF5 Type of hydrocarbon
Peak area of hydrocarbon in sample Peak area of any hydrocarbon in control
C9
7.34
C10
4.87
C11
5.32
C12
3.12
C13
2.86
C14
3.06
C15
3.11
C16
3.03
C17
2.18
C18
1.87
C19
1.72
C20
1.88
C21
1.48
C22
1.22
C23
1.18
C24
1.13
C25
0.86
C26
0.44
C27
0.47
C28
0.39
C29
0
C30
0.46
C31
0.31
C32
0.27
C33
0
C34
0
tendency of crowding towards the periphery. Three different hyphal system were observed: pigmented, branched generative hyphae along with clamp connections and septa; pigmented, thick-walled unbranched skeletal hyphae; and thick-walled rarely branched binding hyphae. These are characteristic of trimitic hyphal system [40]. For molecular identification of the selected fungal strain EF5, genomic DNA was successfully extracted and was subjected to PCR-amplification of the ITS region. The ITS region is used to identify fungi based on conserved sequences in 5.8S, 18S, 28S rRNA, and internal transcribed spacer. Gel electrophoresis of PCR products showed a fragment of about 607 bp. Subsequently, the fragment was sequenced and compared with other sequences to find similarities from GenBank using BLAST program. The results showed the sequences were similar to that of Daedaleopsis sp. (homology 99%; Genbank) [41]. EF5's phylogenetic position was determined in a phylogenetic tree structured using the neighbor-joining method (Fig. 4).
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3.4 | Biodegradation of anthracene and dibenzofuran Table 2 shows the percent of biodegradation of ANT and DBF following incubation with Daedaleopsis sp. According to the results, Daedaleopsis sp. could degrade 75.3 and 69.7 of ANT and DBF, respectively.
3.5 | Optimization of carbon and nitrogen sources on biodegradation of ANT and DBF Different carbon and nitrogen sources were studied to optimize ANT and DBF biodegradation. Figure 5a-d displays the effect of employing various carbon sources on biodegradation of ANT and DBF. According to the results (Figs. 5a and 5b), 10 g L−1 glucose presented improved efficiency compared to sucrose and starch. Furthermore, using 5 g L−1 yeast extract resulted in higher performance of Daedaleopsis sp. in degrading ANT or DBF (Figs. 5c and 5d). However, it seems that ANT was influenced more than DBF by various nitrogen sources. In addition, organic nitrogen sources such as yeast extract and peptone were more effective in ANT biodegradation compared to inorganic nitrogen source, that is, sodium nitrate.
3.6 | Optimization of pH and temperature on biodegradation of ANT and DBF Biodegradation of ANT and DBF by Daedaleopsis sp. in various temperature and pH conditions is shown in Figure 6. Daedaleopsis sp. could degrade ANT and DBF in a relatively wide range of pH from 3 to 9. According to our results, no significant biodegradation was detected in 20 °C. Moreover, Daedaleopsis sp. presented effective degradation from 30 to 60 °C. Interestingly, ANT was degraded more efficiently than DBF in 20–50 °C, and conversely, DBF was degraded more efficiently compared to ANT in 60 °C. In addition, Daedaleopsis sp. showed more competence in biodegrading ANT than DBF in pH 3. The isolated Daedaleopsis sp. strain could degrade 93.7% and 91.2% of ANT and DBF, respectively, in the optimized pH and temperature conditions after 14 days. Daedaleopsis sp. showed the highest biodegradation yield at pH 5 and 40 °C.
3.7 | Asphaltene biodegradation SARA analysis is commonly used to reveal compositional changes in crude oil following alteration [42]. Mass fractions of saturates, aromatics, resins, and asphaltene are presented in Table 3. According to our data (Fig. 7), aromatics content of HCO reduced from 25% to 15.5% and asphaltene content reduced as well from 15% to 1.7%. These changes were concomitant with an increase from 52% to 75% in saturates
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FIGURE 4
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Phylogenetic analysis based on the ITS sequences. The presented tree was derived using the neighbor joining method by MEGA
6.06 software
fraction. Therefore, Daedaleopsis sp. caused a significant reduction of 88.7% in asphaltene and 38% in aromatic fractions following 30 days of incubation in pH 5 and 40 °C. Moreover, an increase of 44.8% in saturates was detected.
Laccase, MnP, and LiP activities in the absence of HCO were 51.3, 145.2, and 214.5 U ml−1, but the presence of HCO increased these enzymes’ activities to 121.5, 231.4, and 352.5 U ml−1, respectively.
3.8 | Ligninolytic enzymes assay in the absence and presence of heavy crude oil
4 | DISCUSSION
Activities of ligninolytic enzymes secreted by Daedaleopsis sp. in presence and absence of HCO are shown in Table 4. According to the results, Daedaleopsis sp. produced laccase, MnP, and LiP but did not produce VP in both conditions.
TABLE 2 Biodegradation of anthracene and dibenzofuran by Daedaleopsis sp. Sample incubated with Daedaleopsis sp.
Percent of degradation
Anthracene (ANT)
75.3
Dibenzofuran (DBF)
69.7
In the present study, a highly potent Daedaleopsis sp. isolate was introduced as an effective agent in biodegradation of toxic and heavy petroleum compounds. There has not been any report on biodegradation ability of Daedaleopsis sp. on heavy petroleum compounds up to now. Daedaleopsis sp. caused a significant decrease in asphaletne content of HCO. Moreover, this isolate demonstrated to be a potent degrader of ANT and DBF in pH 5 and 40 °C. As recalcitrant PAHs pose a great risk in human health, finding powerful organisms effective in PAH's biodegradation is preferred. According to the surface tension analysis performed in the current study, Daedaleopsis sp. could reduce the surface tension of the medium more efficiently (21.6 mN m−1)
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FIGURE 5
Evaluation of the effect of carbon and nitrogen sources on biodegradation of ANT and DBF by Daedaleopsis sp. (a) Effect of carbon source on biodegradation of ANT; (b) effect of nitrogen source on biodegradation of ANT; (c) effect of carbon source on biodegradation of DBF; (d) effect of nitrogen source on biodegradation of DBF. Each data point represents values from three independent experiments ± SEM
compared to other isolated strains. Parach et al. [43] reported a declined surface tension of the medium containing crude oil to from initial 60–26 mN m−1 at the end of incubation. Furthermore, a reduced surface tension (38.3 mN m−1) was also observed after incubation with the yeast Yarrowia lipolytica isolated from Perisan Gulf [44]. Besides, treatment of crude oil with Penicillium sp. RMA1 and RMA2 was reported to reduce the surface tension. According to the published reports, there is a direct relationship between biodegradation ability of fungi and a reduced surface tension [45]. Therefore, Daedaleopsis sp. was selected to conduct the biodegradation experiments.
FIGURE 6
Evaluation of the effect of the pH and temperature on biodegradation of ANT and DBF by Daedaleopsis sp. Effect of various temperatures was assessed in pH 5 and effect of different pHs was evaluated in 40 °C. Each data point represents values from three independent experiments ± SEM. (Control flasks did not show any biodegradation)
Based on the GC/FID analysis of crude oil, Daedaleopsis sp. showed biodegradation activity. In general, lower molecular weight compounds have lower boiling points and exit the GC column in shorter retention times [46,47]. Therefore, longer retention times are indicative of compounds with higher boiling points and shorter retention times are representative of compounds possessing the lower boiling points [46,47]. In accordance with that, fungal treated oil components was detected in shorter retention times than control. Furthermore, results of GC/MS analysis indicated that amounts of C24 hydrocarbons were decreased. This demonstrated that Daedaleopsis sp. could convert longer carbon chain compounds to shorter chain hydrocarbons. This is in line with GC/FID results in our study. Many efforts have been devoted to identify the bacterial or fungal strains capable of utilizing hydrocarbons. However, the identified strains are mostly able to use shorter chain hydrocarbons [48]. Finding a strain capable of degrading longer chain hydrocarbons is of economic value [49]. Employing spore-forming bacterial consortia isolated from Oman oil fields, the researchers have been successful to efficiently transform heavy crude oil into lighter (C11–C27) hydrocarbons following incubation for 21 days. The authors reported the higher ratio of C10–12 hydrocarbons and lower proportion of longer chain hydrocarbons [50]. Furthermore, total degradation of C11–C18 and partial degradation of C19–C24 were reported by Bacillus pumilus [51]. In the current study, Daedaleopsis sp. also presented the capability of biotransforming HCO by decreasing
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TABLE 3 SARA analysis of heavy crude oil fractions following incubation with Daedaleopsis sp.
Fractions
Control (%)
Sample incubated with Daedaleopsis sp. (%)
Saturates
52
75.3 ± 1.27
–
Biodegradation (%)
± 1.53 Aromatics
25 ± 2.2
15.5 ± 1.44
38
Resins
8 ± 0.6
7.5 ± 0.49
6.25
Asphaltenes
15 ± 1.4
1.7 ± 0.26
88.7
All values are means of three independent experiments ± SEM.
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the amount of longer chain hydrocarbons (>C24) and instead increasing the amount of shorter chain hydrocarbons (
