| Microbial Enhanced Oil Recovery(MEOR) is also known as biotechnologically enhanced oil recovery processes, has been proved to have a good potential for application. In this study, six fungal cultures of Aspergillus spp. and three Bacillus strains were isolated from oil-contaminated surface soil samples from an oilfield. The performance of fungi and bacteria to degrade crude oil and wax were investigated using solid enzyme preparations and solid bacterial agents. The aim of the present work was to assess the efficiency of fungal enzyme preparations and solid bacterial agents for crude oil and wax degradation, comprehended mechanism of microbial enhanced oil recovery and determine their feasibility for use in MEOR. The main contributions of the research were summarized as follows:1. Screening and identification of fungal culturesSix pure fungal cultures, designated PJ1 to PJ6, were isolated from the oil-contaminated soil samples. These pure cultures were identified as Aspergillus fumigatus(PJ1, PJ2, PJ3 and PJ5), A. flavus(PJ4), and A. terreus(PJ6) by morphological examination and gene sequencing. r DNA-ITS gene sequences were deposited in the Genbank database under the accession numbers KP 324769 to KP 324774. The six fungal cultures exhibited high efficiency to degrade crude oil and wax after 14 d and 21 d of incubation, and the highest degradation efficiency reached by 79.4% and 38.3%, respectively. These results showed that the fungal cultures had a favorable degrading enzyme system.2. Properties of crude enzyme preparations and proportion tests of crude oil and enzyme solutionWhen grown on the enzyme-producing medium, all fungal cultures produced extracellular enzymes(mixture E1 to E6) with obvious dehydrogenase and catechol 2, 3-dioxygenase activities at 40 °C under aerobic conditions. The properties, proportion tests of crude oil and enzyme solution and performance of fungal extracellular enzymes to degrade crude oil were investigated. The results showed that all of six fungal cultures could produce dehydrogenase and catechol 2, 3-dioxygenase, and the average dehydrogenase activities were 29.68–93.57 mg/g/h, dioxygenase activities were 21.06–25.40 mmol/g/h, and the average dehydrogenase and catechol 2, 3-dioxygenase activity were higher in E5(93.57 mg/g/h and 25.08 mmol/g/h). E1 and E5 can endure salt, acid-base properties and salinity, which show two enzymes have high environment adaptability. Dehydrogenase had dropped significantly when Na Cl and salinity concentration rose to 50 g/L and 92.43 g/L, respectively. Both of two enzyme preparations demonstrated high activity within the 6.0–10.0 range. From using effect and economic considerations, when enzyme solution concentration and proportion of crude oil and enzyme solution are 8 g/L and 1:15, respectively, crude oil removal and emulsibility are generally better in proportion tests of crude oil and enzyme solution.3. Impacts of fungal enzyme preparations on physicochemical properties of crude oil and waxAll the enzyme preparations demonstrated the high efficiency to degrade crude oil and wax. The six enzyme preparations exhibited good degradation efficiencies for alkanes, aromatics, resins and asphaltenes, the degradation rate of resins and asphaltenes reach up to 35.6% and 34.6% for contained in the crude oil sample. Enzyme preparations caused progressive increases in the total number of gasifiable n-alkanes. Correspondingly, the total peak area of gasifiable n-alkanes was increased by 5.6% to 28.8%. All of the fungal enzyme preparations were able to decrease crude oil viscosity by nearly half, i.e., 40.5%–59.0%. Enzymatic degradation of crude oil was accompanied by dynamic production of gases(CO2 and H2) and organic acids(oxalate and propionate). The CO2 and H2 production rate were 21.3%–53.3% and 38.3%–100%, respectively. The total acid number ranged from 1335 to 1448 mg/L, total acidity ranged from 22.25–24.13 mmol/L, while the p H of the degradation solutions decreased by 22.7%–29.4% compared to the control. Additionally, the enzyme preparations removed 83.40%–87.78% of crude oil adsorbed on filter paper, that is, 7.41–7.80-fold that of the control. All the enzyme preparations also demonstrated the high efficiency to degrade wax. Compared with the control, enzymatic degradation significantly changed the solubility of paraffin wax in n-hexane. The soluble ratio of wax, SW% values of enzymatically-degraded samples reached 78.6%–85.1%, with 31.9%–44.6% increases compared to the control. Enzyme preparations caused progressive decrease in the light hydrocarbon content, i.e., 3.7%–26.9%, and increase in the heavy hydrocarbon content, i.e., 5.5%–19.6%. The temperature of the phase transition from solid to liquid increased 4 ℃. The process of wax degradation by crude Aspergillus enzyme preparations was accompanied by dynamic production of gases, mainly CO2 and H2. The CO2 and H2 production rate were 26.7%–66.7% and 50.0%–125.0%, respectively. The total acid number of the reaction solutions was within the range of 1286.4–1395.0 mg/L, total acidity ranged from 21.44–23.25 mmol/L, while the p H measurements decreased by 22.3%–28.5% compared to the control. The shape of wax crystals was obviously changed due to enzymatic degradation, with a rough surface and a loose structure compared to the control.4. Identification and characterization of bacterial culturesThree pure bacterial cultures, designated 5-2a, 6-2a, and 6-2c were isolated from the oil-contaminated soil and could utilize crude oil as a carbon source. Based on r RNA sequencing analysis, these pure cultures were identified as Bacillus atrophaeus(5-2a), B. aryabhattai(6-2a) and B. amyloliquefaciens(6-2c). Solid bacterial agents from Bacillus strains 5-2a, 6-2a, and 6-2c were respectively named B1(B. atrophaeus 5-2a), B2(B. aryabhattai 6-2a) and B3(B. amyloliquefaciens 6-2c). All of three pure bacterial cultures are aerobic, motionless, and can survival within 30-80 temperature and 0~100 g/L salinity rang. The results indicated that three cultures were resistant to high temperature high salinity environment. The three bacterial cultures exhibited varying degradation efficiencies after 7 d of incubation, i.e., 30.0%–57.8% for alkanes, 47.4%–73.1% for aromatics, 61.4%–72.8% for resins, and 42.1%–57.9% for asphaltenes contained in the crude oil sample. Bacterial degradation caused progressive increases in the light hydrocarbon, i.e., 27.4% to 61.0%, and then decreasing crude oil viscosity, i.e., 7.4%–11.1%. Additionally, the fermentation broth removed 66.5%–94.2% of crude oil adsorbed on filter paper, that is, 5.32–7.53-fold that of the control.5. Impacts of bacterial agents on physicochemical properties of crude oil and waxAll of bacterial agents demonstrated the high efficiency to degrade crude oil and wax. Three bacterial agents exhibited varying degradation efficiencies after 4 d of incubation, i.e., 12.5%–15.1% for alkanes, 23.9%–39.3% for aromatics, 19.8%–24.2% for resins, and 53.1%–56.2% for asphaltenes contained in the crude oil sample. Bacterial agents caused progressive increases in the total number of gasifiable n-alkanes. Correspondingly, the total peak area of gasifiable n-alkanes was increased by 62.6%–108.0%. All of bacterial agents were able to decrease crude oil viscosity by 24.7%–29.4%. Bacterial degradation of crude oil was accompanied by dynamic production of gases(CO2 and H2) and organic acids(oxalate and propionate). The CO2 and H2 production rate were 74.1%–81.0% and 138.3%–152.3%, respectively. The total acid number ranged from 1410 to 1560 mg/L, total acidity ranged from 23.5–26.0 mmol/L, while the p H of the degradation solutions decreased by 26.5–36.0% compared to the control. Laboratory studies had shown that all the three bacterial agents from Bacillus sp. efficiently improved the solubility of wax in n-hexane and degraded n-alkanes in wax. The soluble ratio of wax reached 68.6%–77.2%, with 15.0%–29.5% increases compared to the control. Bacterial agnts caused progressive decrease in the light hydrocarbon content, i.e., 6.9%–14.9%, and increase in the heavy hydrocarbon content, i.e., 18.4%–25.7%. The process of wax degradation by bacterial agents was accompanied by dynamic production of gases, mainly CO2 and H2. The CO2 and H2 gas production rate were 65.0%–80.0% and 120.0%–150.0%, respectively. The total acid number of the reaction solutions was within the range of 1373–1545 mg/L, total acidity ranged from 22.88–25.75 mmol/L, while the p H measurements decreased by 27.3%–36.8% compared to the control. The shape of wax crystals was obviously changed due to bacteria degradation, with a rough surface and a loose structure compared to the control.6. Effect of microbial cleaning and prevention waxThree pure bacterial cultures demonstrated the high efficiency to degrade wax. Compared with the control, bacterial degradation efficiently improved the solubility of wax in n-hexane. The soluble ratio of wax reached 57.7%–62.0%, with 1.5%–9.0% increases compared to the control. Fermentation broth removed 36.2%–100.0% of wax adsorbed on glass sheet. Three bacterial cultures showed high efficiency to clean and prevent wax, and rate of prevention wax reached 94.64%–98.10%, rate of cleaning wax reached 69.1%–89.3%. Additionally, three bacterial cultures are prone to be adhered to the surface of solid material that would help to wax deposition. Adhesion density of glass and steel disc surface reached 1.95×106–7.67×108 CFU/cm2 and 8.67×104–7.48×108 CFU/cm2, respectively, cell in loose form adhered to glass and steel disc surface reached 3.22×104–6.81×106 CFU/cm2 and 6.67×103–2.00×108 CFU /cm2, respectively.7. Production of lipopeptide biosurfactant by Bacillus atrophaeus 5-2a and its crude oil removalBacillus atrophaeus 5-2a can produce biosurfactants by fermenting cheaper raw materials Urea. The biosurfactant showed excellent surface active potential, diameter of oil spreading, emulsification index and surface tension value reached 19.1 cm, 59.49% and 25.43 m N/m, respectively, and crude biosurfactant yield reached 0.77 g/L. The biosurfactants produced by 5-2a were quite stable over a wide range of p H, salinity and temperatures, i.e., temperatures from 20 to 100 °C, salinity from 10 to 90 g/L, and p H from 6 to 13. The biosurfactants removed 90.0%–93.1 % of crude oil adsorbed on sand and filter paper. The results are indicative of the great potential of the culture for applications in enhanced crude oil recovery.8. Mechanisms of impacts of fungal enzyme preparations and bacterial agents on physicochemical properties of crude oil and waxThrough a complexity of enzymatic processes, crude oil and wax were degraded by microbes catalyzed by enzyme. The mechanisms by which impacts processes operate can be quite complex and may involve multiple biochemical process steps:(1) biodegradation, fungal enzyme preparations and bacterial agents efficiently degraded wax and various oil fractions, i.e., alkane, aromatic, resins, asphaltene, and decreased the viscosity of crude oil to improve crude oil mobility and change shape of wax crystals;(2) gases production, the process of crude oil and wax degradation by fungal enzyme preparations and bacterial agents was accompanied by dynamic production of gases, mainly CO2 and H2. Such gases produced in situ can contribute to pressure build-up in a pressure-depleted reservoir. These gases may dissolve in the crude oil and reduce its viscosity;(3) acids production, in addition to the gases, substantial acids were produced during wax and crude oil degradation by fungal enzyme preparations and bacterial agents, the organic acids mainly include oxalate and propionate. These acids can dissolve the carbonate rock, thereby increasing its permeability and porosity, moreover, they dramatically reduces the interfacial tension and form an oil-in-water emulsion, thereby mobilizing this trapped oil by increasing the capillary number; and(4) role of biosurfactants, six fungi and three bacteria can produce biosurfactants. Surfactants thus contribute positively to improve oil recovery by reducing IFT and also by altering the wettability of reservoir rock for water-flood to displace more oil from the capillary network. Surfactants enable emulsify paraffin efficiently and remove paraffin-based skin damage from the well bore.9. Mechanisms of microbial cleaning and prevention waxMicrobial remediation utilizes microbes or their metabolic byproducts(e.g. surfactants and paraffin solvents) to prevent and remove paraffin damage. The mechanisms involve three points:(1) biodegradation, three bacteria demonstrated effectively paraffin wax degradation, decomposing heavy wax fractions into lighter fractions, which decreased wax precipitation;(2) formation of biofilms, three bacteria can more substantively and firmly adsorbed on metal surface and form biofilms. Biofilms are heterogeneous systems of bacteria that can prevent paraffin deposition on suface of metal;(3) synthesis of surfactant, surfactant can improve wettability by adsorbing on metal surface, thereby preventing paraffin precipitation; surfactants enable emulsify paraffin efficiently and remove paraffin-based skin damage from the well bore. |
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