The ability of strains HJ2 and HJ4 to degrade heavy oil was initially tested by the growth of cultures on oil and bitumen plates of mineral salts medium (containing 20 g L−1 heavy oil or bitumen), respectively (Fig. 1). The biodegradation of heavy oil was verified by observation of a decrease in the total weight of the heavy oil and a redistribution of its fractions (saturates, aromatics, resins, and asphaltenes). Table 1 showed that there was a significant difference (P < 0.05) between the control group and the treated samples in the total petroleum hydrocarbons. The degradation ratios of total petroleum hydrocarbons (15 d) for the fungi HJ2 and HJ4 were 25.29% and 27.36%, respectively. After 15 d of aerobic degradation saturates and asphaltenes were decreased by 36.19–39.08% and 7.92–10.89%, respectively, while the aromatics and resins were increased by 2.70–5.02% and 16.00–18.00%, respectively (Table 1). GC analysis 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. The degradation rates of n-alkanes with different chain lengths were determined from the decreases in the peak areas of individual n-alkanes. Strain HJ4 exhibited higher degradation capacity than HJ2, and particularly showed higher degradation efficiency of long-chain n-alkanes (>37.00 min retention time), although the two strains had similar overall biodegradation rates of heavy oil (Table 2).
Generally, mesophilic microorganisms most 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 the heavier fractions of resin and asphaltene [22, 23]. In our study, A. terreus HJ2 and A. nidulans HJ4 showed the ability to degrade long alkyl chains and asphaltenes of heavy oil in aerobic conditions. Microbial degradation of heavy oil is a complex process that involves various types of enzymes, such as oxygenases, dehydrogenases and hydroxylases. These enzymes can catalyze aromatic and aliphatic hydroxidation, oxidative deamination, hydrolysis, and other biochemical transformations of substances in the original oil . A test of in-vitro oil degradation by the crude enzymes demonstrated an accelerated biodegradation of aliphatic, aromatic, resin, and asphaltene fractions due to enhanced enzyme activities in the enzymatic 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 hydrocarbon degradation activity
When grown on the enzyme-producing medium, strains HJ2 and HJ4 produced extracellular enzymes with dehydrogenase and C23O activities; the enzyme mixtures were designated E2 and E4, respectively. The average dehydrogenase activity of the extracellular enzymes was 66.76 ± 0.25 (E2) and 68.77 ± 0.18 (E4) µg g−1 min−1. The C23O activity was 3.65 ± 0.014 (E2) and 4.32 ± 0.028 µmol min−1 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 . The enzyme preparations E2 and E4 are capable of degrading hydrocarbons and releasing biogas. Fig. 4 showed the cumulative gas production after 15 d with 2 g of hydrocarbon as the enzymatic substrate. During degradation, the maximum gas values for 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 showed higher gas production than E2, which implied higher degradation ability of hydrocarbons by E4 as compared to E2. Both enzyme mixtures showed higher efficacy in biodegrading wax and light oil than bituminous crude oil. According to Al-Sayegh et al. , due to being rich in resin and asphaltene, 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 enzyme mixtures E2 and E4. Based on the gas production results, which measure enzymatic hydrocarbon degradation activity, we inferred that the enzyme mixtures 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 saturates 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 control levels, 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 with the control level. The observed trends in the four fractions of heavy oil indicate that enzyme concentration is a critical factor in determining the oil degradation rate.
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 hydrocarbon and resin fractions of heavy oil were relatively increased after degradation. According to Mohamed et al. , the primary consumption or depletion of the saturated fraction can result in high polyaromatic and asphaltene content, which are more recalcitrant to biodegradation. In MEOR, it is important to select appropriate strains that can survive and secret specific metabolic products under the reservoir conditions, yet it remains difficult to isolate microorganisms that can tolerate the geological 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 accompanied by 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 indicated that these gases were mainly CO2 and H2 with small amounts of methane. Production of gas showed significant correlations with the amount of enzyme (P < 0.05). The correlation coefficients were 0.93984 and 0.99569, respectively. The gas produced by enzymatic degradation can contribute to enhanced heavy oil recovery by increasing the pressure of the reservoir and reducing oil viscosity.
Effect of surfactants 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 . The effects of surfactants of both biological and chemical origin on the enzymatic degradation of heavy oil by E2 and E4 were measured in degradation assays. 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 saturates was decreased to 1327 mg (E2), 1353 mg (EB2), 1321 mg (EC2), 1294 mg (E4), 1371 mg (EB4), and 1272 mg (EC4), respectively; the content of aromatics was increased to 417 mg (E2), 352 mg (EB2), 330 mg (EC2), 440 mg (E4), 372 mg (EB4), 371 mg (EC4), respectively; the content of asphaltenes was slightly decreased to 93–97 mg; and the resins were increased to 181–233 mg by the two enzymes but decreased from 147–104 mg if surfactants were also added. The biogas yield was reduced from 67.0 to 50.0 mL bottle−1 and 86.5 to 56.5 mL bottle−1, respectively, for crude enzyme preparations E2 and E4 on addition of the biosurfactants and SDS (Table 4). The results show that addition of surfactant had an inhibitory effect on the enzymatic degradation of heavy oil. It has been reported that many surfactants are antimicrobial, and the antimicrobial activity depends on the surfactant concentration and the strain used for biodegradation . Sajna et al.  and Tian et al.  reported that most surfactants exhibit toxicity to microorganisms at above 1 critical micelle concentration, which influences cell surface properties and metabolic pathways, then results in a decrease in the crude oil biodegradation rate.
Enzymatic degradation of gasifiable n-alkanes
Enzymatic degradation had a significant effect on the relative quantities of gasifiable n-alkanes compared with controls. 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, and 11–13 new fractions appeared, while three fractions disappeared relative to the control. 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 efficacy for long-chain alkanes. The increase of peak area of individual n-alkanes was 11.46 to 198.19% (E2) and 15.16 to 537.41% (E4). This variation of the oil components, especially the decrease of long-chain fractions of saturated hydrocarbons, 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 . It has been reported that numerous functional microbes (e.g., Petrobacter sp., Enterobacter sp., Bacillus sp., and Geobacillus sp.) can produce highly degradative enzymes, resulting in the breakdown of hydrocarbon chains, making them lighter and enhancing oil mobility [34, 35]. In the present study, the two tested fungal enzyme preparations showed strong abilities to degrade heavier fractions into lighter fractions in oxygen-deprived conditions. Although the exact mechanism is not known, the fungal enzymes might specifically shorten long chain hydrocarbons, depolymerize asphaltenes and increase the solvent fraction, which would benefit heavy oil recovery.
Viscosity reduction of heavy oil
High viscosity is an important factor responsible for poor heavy oil recovery . Conventional methods for reducing oil viscosity, such as heating, emulsification, and dilution with light crude oil, have the disadvantages of high cost in application and potential damage to the formation. In this study, the potential use of fungal enzyme preparations to reduce oil viscosity was, therefore, examined. The two fungal enzyme preparations were able to reduce the viscosity of heavy oil from 29,700 to 13,700, and 10,000 mPa s, respectively. E4 had greater viscosity reduction efficiency (66.33%) than E2 (53.87%). Previous 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 with these bacteria, the fungal enzyme preparations exhibited markedly higher abilities to decrease heavy oil viscosity in the current study. Moreover, enzyme preparations have the advantage of short heavy oil degradation time and they do not require the use of live microorganisms, which is conducive to use 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., asphaltene), leading to a decrease of average molecular weight and reduction of heavy oil viscosity.
Removal of heavy crude oil from sand
Table 5 shows the amount of heavy oil removed from artificially contaminated sand after treatment with crude enzyme solution. The removal efficiencies of the two crude enzyme solutions (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 fungi 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 known as a promising agent for enhancing oil recovery, have been successfully applied in oilfield exploitation [38, 39]. França et al.  obtained that the cell-free fermented broth of B. subtilis ICA56 containing biosurfactant removed 85% of crude oil, which highlighted the crucial role of biosurfactant for the cleaning process. Thus, the crude enzyme solutions (low content of biosurfactants) were insufficient to mobilize a significant amount of entrapped oil.
The removal effects of the two crude enzyme solutions were possibly due to heavy component degradation and gas production. The interactions of gas with oil, as well as the bioconversion 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 14.7% 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 by degrading heavy oil fractions . Our results suggest that supplementation with biosurfactants after enzyme solution flooding may be an more effective strategy to improve heavy oil recovery than pure enzyme solution flooding.