Screening and identification of oil-degrading fungi
Four pure fungal strains with the ability to degrade heavy oil, designated HJ1 to HJ4, were isolated from bitumen enrichment cultures. Strains HJ2 and HJ4 exhibited good growth on oil plates of mineral salts medium (Fig. 1). Thus, we selected these two strains as potential candidates for analyses of heavy oil biodegradation, extracellular enzyme production, and enzymatic degradation of heavy oil. Based on morphological analysis (Fig. 2) and partial sequencing of the rDNA internal transcribed spacer (ITS) region (Fig. 3), strains HJ2 and HJ4 were identified as Aspergillus terreus and A. nidulans, respectively. The rDNA-ITS gene sequences of the two strains were deposited in the GenBank database with accession numbers MG 732935 and MG 732936. Several fungi of the genus Aspergillus isolated from petroleum-contaminate dtropical soils are known to be able to degrade petroleum hydrocarbons. They can use petroleum hydrocarbons as their sole carbon and energy source to produce simpler compounds such as CO2 and H2O .
Fungal degradation of heavy oil
The ability of A. terreus HJ2 and A. nidulans HJ4 to degrade heavy oil was initially tested on oil and bitumen agar plates of mineral salts medium (containing 20 g L−1 heavy oil or bitumen; Fig. 1). The biodegradation of heavy oil was verified by observation of a decrease in the total weight of heavy oil and a redistribution of four fractions (alkans, aromatics, resins, and asphaltenes). After 15 d of fungal degradation, there was a significant difference in the total weight of heavy oil between the control group and fungi-treated samples (P < 0.05). The degradation efficiencies of heavy oil by strains HJ2 and HJ4 were similar, 25.29% and 27.36%, respectively. Among the four fractions, alkanes were decreased by 36.19–39.08%, while resins were increased by 16.00–18.00% (P < 0.05; Table 1). Gas chromatography (GC) provided a more detailed assessment of the heavy oil degradation and characterized the variation of saturates before and after fungal degradation. Both strains HJ2 and HJ4 were able to degrade the heavy oil efficiently; 33.33–66.67% of the n-alkanes were eliminated when compared to the control group. The degradation efficiencies of n-alkanes with different chain lengths were determined from the decreases in the peak areas of individual n-alkanes. Strain HJ4 exhibited a higher degradation capacity for n-alkanes and particularly showed higher degradation efficiencies of long-chain n-alkanes (retention time >37.00 min) compared to HJ2 (Table 2).
Generally, mesophilic microorganisms readily degrade alkanes with carbon chain lengths ranging from 10 to 16 to generate energy for their growth; however, polycyclic aromatic hydrocarbons, which are less bioavailable than alkanes, are not completely degraded because they are more effectively partitioned into heavier fractions (e.g., resins and asphaltenes) [22, 23]. Here, we found that A. terreus HJ2 and A. nidulans HJ4 had the ability to degrade long alkyl chains of heavy oil in aerobic conditions. Microbial degradation of heavy oil is a complex process involving various enzymes such as oxygenases, dehydrogenases, and hydroxylases; the enzymes can catalyze biochemical transformations including aliphatic and aromatic hydroxidation, oxidative deamination, and hydrolysis of substances in heavy oil . A test of in-vitro oil degradation by crude enzymes demonstrated an accelerated biodegradation of aliphatic, aromatic, resin, and asphaltene fractions due to enhanced enzyme activities in the co-culture . Thus, we speculated that our Aspergillus isolates HJ2 and HJ4 produce multiple enzymes that can be directly used to degrade heavy oil.
Enzymatic degradation of heavy oil
Enzymatic hydrocarbon degradation
When grown on the enzyme-producing medium, both strains HJ2 and HJ4 produced extracellular enzymes with dehydrogenase and catechol 2,3-dioxygenase (C23O) activities. The enzyme mixtures of strains HJ2 and HJ4 were designated E2 and E4, respectively. Their average dehydrogenase activities were 66.76 ± 0.25 (E2) and 68.77 ± 0.18 (E4) µg g−1 min−1; the average C23O activities were 3.65 ± 0.014 (E2) and 4.32 ± 0.028 (E4) U mg−1. Dehydrogenase and C23O activities reflect the ability of microorganisms to degrade alkanes and aromatic hydrocarbons. The activities of the two fungal enzyme preparations suggested that they could be used directly for hydrocarbon degradation.
The chemical inertness of hydrocarbons poses an energetic and mechanistic challenge for microbial metabolism. This is particularly true for the initial activation and eventual cleavage of the apolar C–H bond, where high energy barriers must be overcome, and, therefore, the rate of conventional biodegradation of hydrocarbons is relatively slow . In the present study, the enzyme preparations E2 and E4 were capable of degrading hydrocarbons and releasing biogas. The cumulative gas production after 15 d of enzymatic degradation with 2 g of hydrocarbons as the substrate is shown in Fig. 4. The maximum gas productions from bituminous crude oil (15% w/w), heavy oil, light oil, wax, and liquid paraffin were 40.0–67.5, 64.0–80.5, 75.0–80.0, 81.0–109.5, and 73.0–76.0 mL bottle−1, respectively, for E2 and E4. E4 gave a higher gas production than E2 when applied in the same quantities; this implied the greater ability of E4 to degrade petroleum hydrocarbons compared to E2. Both enzyme preparations showed higher efficiency in biodegrading wax and light oil than bituminous crude oil. According to Al-Sayegh et al. , due to being rich in resins and asphaltenes, bituminous crude oil is relatively resistant to biodegradation and more slowly degraded than linear oils. The aliphatic fraction, which is more susceptible to degradation than the aromatic and asphaltene fractions, was probably more easily broken down by the preparations E2 and E4. Based on the gas production results, we inferred that the enzyme preparations E2 and E4 can be used to enhance oil mobilization by degrading heavy oil and producing bioproducts.
Effect of enzyme concentration on heavy oil degradation
Evaluation of heavy oil degradation by different concentrations of crude enzymes in oxygen-deprived conditions showed positive results (Table 3). The four fractions of heavy oil were redistributed after 15 d of incubation at 40 °C. As the crude enzyme concentration increased from 2% to 10%, the content of alkanes markedly decreased, with the percentages of degradation in the range of 16.28–27.61%. The content of aromatics and resins first increased and then decreased, both of which remained higher than the values of the control group, by 30.85–64.89% and 2.45–28.82%, respectively. By contrast, the content of asphaltenes decreased slightly by 2.97–13.86% compared to the values of the control group. The observed trends in the four fractions of heavy oil indicate that enzyme concentration is a critical factor in determining the oil degradation efficiency.
The degradation efficiency of different heavy oil fractions by bacterial consortia was reported to be 10.57–23.68% for alkanes, 6.03–20.62% for aromatics, and 3.63–16.90% for resins and asphaltenes [10, 26, 27]. By comparison, the fungal enzyme preparations from our Aspergillus cultures had higher abilities to degrade saturates, while the aromatic and resin fractions of heavy oil were relatively increased after enzymatic degradation. According to Mohamed et al. , the primary consumption or depletion of the saturated fraction can result in high content of polyaromatics and asphaltenes, which are more recalcitrant to biodegradation. In MEOR, it is important to select appropriate strains that can survive and secret specific metabolites under the reservoir conditions, yet it remains difficult to isolate functional microorganisms that can tolerate the harsh environment of oil reservoirs. Therefore, the fungal enzyme preparations, which exhibit unique biodegradation capabilities and require no use of live microorganisms, are a good choice for MEOR.
The process of heavy oil degradation by the two enzyme preparations at different concentration was associated with dynamic production of gas. The total gas production varied from 25.5 to 100.0 mL bottle−1 for preparation E2 and 24.0 to 186.5 mL bottle−1 for E4 (Table 3). GC analysis showed that these gases were mainly CO2 and H2 with small amounts of CH4. Production of gas was significantly correlated with the concentration of enzyme preparation added (P < 0.05); the respective correlation coefficients were 0.93984 (E2) and 0.99569 (E4). The result indicates that when applied within the range 0–10%, a higher concentration of the enzyme preparation would give a higher production of gas from heavy oil. The gas produced by enzymatic degradation can contribute to enhanced heavy oil recovery by increasing the pressure of the reservoir and reducing the viscosity of the oil.
Effect of surfactant and biosurfactant on heavy oil degradation
Surfactants are amphiphilic compounds which partition at the interface between fluid phases with various polarities. Surfactants decrease interfacial and superficial tensions of solutions and facilitate the bioavailability of hydrophobic chemicals by solubilization and emulsification . In the present study, we evaluated the effects of surfactants of both biological and chemical origin on the enzymatic degradation of heavy oil by crude enzymes (Table 4). In the control group (without crude enzyme solution), gravitational analysis of 2 g heavy oil showed a content of: saturates, 1567 mg; aromatics, 274 mg; resin, 157 mg; and asphaltene, 101 mg. After 15 d of enzymatic degradation, the content of alkanes was substantially decreased to 1327–1353 mg (E2 with or without bacterial biosurfactant and sodium dodecyl sulfate [SDS]) and 1272–1371 mg (E4 with or without biosurfactant and SDS); the corresponding content of aromatics was considerably increased to 330–417 mg and 371–440 mg; the content of asphaltenes was slightly decreased to 95–97 mg and 93–97 mg; and the content of resins was increased to 181–202 mg by the crude enzymes but decreased to 147–104 mg if (bio)surfactants were also added. For crude enzyme preparations E2 and E4, the biogas yield was reduced from 67.0 to 50.0 mL bottle−1 and 86.5 to 52.0 mL bottle−1, respectively, on addition of the (bio)surfactants. The results show that addition of surfactant and biosurfactant had an inhibitory effect on the enzymatic degradation of heavy oil. Many surfactants reportedly have antimicrobial properties or inhibitory effect on key enzymes, depending on the surfactant concentration and the microbial strain used for biodegradation [30, 31]. Specifically, the surfactants exhibit toxicity to microorganisms and enzymes at above the critical micelle concentration, which changes cell surface properties, alters native enzyme protein conformation, and influences metabolic pathways, resulting in a decrease in the biodegradation of crude oil [31–33].
Enzymatic degradation of gasifiable n-alkanes
Enzymatic degradation had a significant effect on the relative quantities of gasifiable n-alkanes compared to the control group. More detailed assessment of the variation of n-alkanes can be found in Table 2. The two enzyme preparations caused apparent increases in the total number of gasifiable n-alkanes; 11–13 new fractions appeared, while three fractions disappeared relative to the control group. Correspondingly, the overall total peak area of gasifiable n-alkanes was increased by 91.77% (E2) and 130.73% (E4). Both enzyme preparations showed higher degradation efficiency for long-chain alkanes. The increase in the peak area of individual n-alkanes ranged from 11.46 to 198.19% (E2) and 15.16 to 537.41% (E4). This variation of oil components, especially the decrease of long-chain alkanes, has positive effects on the physicochemical properties of the heavy oil, such as light component increase, viscosity reduction, and fluidity enhancement.
Degradation of heavy fractions is considered one of the main mechanisms of MEOR by which the oil’s viscosity and freezing point are reduced, which in turn will increase the oil’s mobility in situ . Numerous functional microbes, such as members of Petrobacter, Enterobacter, Bacillus, and Geobacillus, can produce highly degradative enzymes, resulting in the breakdown of hydrocarbon chains, making them lighter and enhancing oil mobility [35, 36]. In the present study, the two fungal enzyme preparations from Aspergillus spp. showed strong abilities to degrade heavy oil into lighter fractions in oxygen-deprived conditions. Although the exact mechanism is still unknown, the fungal enzymes might specifically shorten long chain hydrocarbons, depolymerize asphaltenes, and increase the solvent fraction, which would facilitate heavy oil recovery.
Viscosity reduction of heavy oil
High viscosity is a crucial factor responsible for poor heavy oil recovery . Conventional methods for reducing oil viscosity include heating, emulsification, and dilution with light crude oil, which have the disadvantages of high cost in application and potential damage to the formation . Therefore, we examined the potential use of fungal enzyme preparations to reduce oil viscosity in the present study. The two fungal enzyme preparations with a final concentration of 4% (w/v) were able to reduce the viscosity of heavy oil from 29,700 to 13,700 (E2) and 10,000 (E4) mPa s. This result showed that E4 had greater viscosity reduction efficiency (66.33%) than E2 (53.87%). Other studies have shown that some bacterial strains (e.g., Bacillus subtilis, B. licheniformis, and Geobacillus stearothermophilus) reduce heavy oil viscosity by 15.47–40.06% under aerobic and aerobic conditions [26, 27]. Compared to these bacteria, the fungal enzyme preparations tested in the current study exhibited markedly higher abilities to reduce heavy oil viscosity. Moreover, enzyme preparations have the advantage of short degradation time and they do not require the use of live microorganisms, which is conducive to application in heavy oil reservoirs with poor growth of oxygen-consuming microorganisms in situ. The high contents of long-chain hydrocarbons with a complex structure, heteroatoms (e.g., O, N, S, and metals), and asphaltenes in heavy oil lead to its high viscosity . Moreover, these adverse conditions usually have negative effects on biodegradation of the oil. However, the fungal enzyme preparations used in our study exhibited the ability to decompose long-chain alkanes and heavy fractions (e.g., asphaltenes), leading to a decrease of average molecular weight and reduction of heavy oil viscosity.
Removal of heavy crude oil from sand
Table 5 lists the results of heavy oil removal from artificially contaminated sand after treatment with crude enzyme solution. The removal efficiencies of the two enzyme preparations (E2 and E4) ranged from 7.85 to 8.48%, that is, 2.29–2.47-fold that of the control (3.43%). Fig. 5 shows the removal efficiency of heavy oil adsorbed on sand by the crude enzyme solution. The fungal isolates in our study showed lower ability to produce biosurfactants than some bacteria (e.g., B. subtilis and P. aeruginosa). The diameter of oil spreading from the crude enzyme solutions reached only 12.0–13.0 cm (the oil spreading experiment was carried out according to the method described by Zhang et al. ). Biosurfactants are emerging as a promising agent for enhancing oil recovery and have been successfully applied in oilfield exploitation [39, 40]. França et al.  showed that the cell-free fermented broth of B. subtilis ICA56 containing biosurfactant removed 85% of crude oil from contaminated sand, which highlighted the crucial role of biosurfactant for the cleaning process. Thus, the crude enzyme solutions with relatively low content of biosurfactants might be insufficient to mobilize a significant amount of entrapped oil.
The removal of heavy oil by the two crude enzyme solutions was possibly due to heavy component degradation and gas production. The interactions of gas with oil, as well as the biotransformation of heavy oil fractions to lighter fractions, are mechanisms responsible for increasing the mobility and recovery of heavy oil . Xia et al.  reported that one enriched methanogenic consortium increased by 14.70% of the tertiary enhanced oil recovery by oil degradation and methane production in core flooding tests, and the gas production made the inner pressure of the microbial core holder increase from 0.2 to 20.45 MPa; the viscosity of the heavy oil was reduced by 72.45%. Biodegradation in oil reservoirs affects the quantity and quality of the crude oil; crude oil will become lighter and more valuable through degradation of heavy oil fractions . Our results suggest that supplementation with biosurfactants after enzyme solution flooding may be a more effective strategy to improve heavy oil recovery compared to flooding with enzyme solution only.