CN109486775B - Fungal enzyme for increasing content of gasifiable oil in crude oil and preparation method and use method thereof - Google Patents
Fungal enzyme for increasing content of gasifiable oil in crude oil and preparation method and use method thereof Download PDFInfo
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- 238000002156 mixing Methods 0.000 claims description 5
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention discloses a fungal enzyme for improving the content of gasifiable oil in crude oil, a preparation method and a use method thereof, wherein the fungal enzyme comprises the following raw materials: an oil displacement fungus and an enzyme production culture medium; wherein the enzyme-producing medium comprises wheat bran, salt solution and crude oil. The invention utilizes the fungal enzyme to carry out enzymatic conversion on crude oil, can degrade and convert high molecular components including asphaltene in the crude oil into micromolecular vaporizable components, and obviously improves the content of saturated hydrocarbon and aromatic hydrocarbon in enzymatic conversion products of the crude oil; the preparation method is simple and easy to operate and implement, can provide theoretical support for improving the quality of crude oil by using fungal enzyme, and simultaneously provides scientific basis for improving the process design of gasoline and low-boiling-point coal and diesel oil yield when crude oil is refined by using fungal enzyme conversion.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a fungal enzyme for improving the content of gasifiable oil in crude oil, and a preparation method and a use method thereof.
Background
Gasoline, kerosene and diesel oil are gasifiable oil in crude oil, and their main components are saturated hydrocarbon and partial aromatic hydrocarbon, and their boiling ranges are 34-205 deg.C, 180-310 deg.C and 180-370 deg.C respectively. The gasifiable oil in the invention refers to gasoline which can be gasified at 230 ℃ in crude oil and part of low-boiling kerosene and diesel oil. Gasoline, kerosene and diesel are blood of industry and transportation industry, and are closely related to human life. Under the condition of increasingly depleted petroleum resources, the method for improving the utilization rate of crude oil is one of important ways for solving the shortage of petroleum resources. If the high molecular components in the crude oil can be converted into small molecular vaporizable components, the yields of gasoline, part of low-boiling kerosene and diesel oil during crude oil refining can be improved, and the resource utilization rate and economic benefit of the crude oil are further improved.
Biological enzymes are proteins used to catalyze various biochemical reactions. Harris and McKay propose the use of enzymes for crude oil and natural gas industry desulfurization and biopolymer pre-processing. Recent researches show that the fungus extracellular enzyme can degrade macromolecules such as asphaltene, paraffin and the like in crude oil into small molecules under the condition of normal temperature and normal pressure, so that the viscosity of the crude oil is greatly reduced. However, the conversion rate of the fungal enzymes to degrade macromolecular components in crude oil into small-molecular vaporizable components is not clear, and whether or not it can convert heavy components such as asphaltenes into saturated hydrocarbons has yet to be confirmed.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide the fungal enzyme for improving the content of gasifiable oil in crude oil, the preparation method and the use method thereof, and the fungal enzyme is utilized to carry out enzymatic conversion on the crude oil, so that high molecular components including asphaltenes in the crude oil can be degraded and converted into small molecular gasifiable components, and the content of saturated hydrocarbon and aromatic hydrocarbon in enzymatic conversion products of the crude oil is obviously improved; the preparation method is simple and easy to operate and implement, can provide theoretical support for improving the quality of crude oil by using fungal enzyme, and simultaneously provides scientific basis for improving the process design of gasoline and low-boiling-point coal and diesel oil yield when crude oil is refined by using fungal enzyme conversion.
In order to achieve the above purpose, the present invention is realized by the following technical scheme.
(one) a fungal enzyme that increases the content of gasifiable oil in crude oil, comprising the following raw materials: an oil displacement fungus and an enzyme production culture medium; wherein the enzyme-producing medium comprises wheat bran, salt solution and crude oil.
Preferably, the displacing fungus comprises Aspergillus oryzae Z3, aspergillus spelunceus Z05, aphanocladium aranearum Z06 or Aspergillus sydowii Z10.
Preferably, the inoculation amount of the oil displacement fungi is 0.1% -0.5%.
Preferably, the salt solution comprises potassium nitrate, potassium dihydrogen phosphate, anhydrous magnesium sulfate, and water.
Preferably, the enzyme-producing medium comprises 7-13 parts of wheat bran, 5-11 parts of salt solution and 0.5-2.0 parts of crude oil.
Preferably, the salt solution comprises 2-8 parts of potassium nitrate, 0.2-2 parts of monopotassium phosphate, 0.1-0.9 parts of anhydrous magnesium sulfate and 1000 parts of water.
(II) a method for preparing fungal enzyme for increasing the content of gasifiable oil in crude oil, comprising the following steps:
step 1, mixing potassium nitrate, monopotassium phosphate, anhydrous magnesium sulfate and water to obtain a salt solution;
step 2, uniformly mixing the salt solution with wheat bran and crude oil, and sterilizing to obtain an enzyme-producing culture medium;
and step 3, inoculating the oil displacement fungi into the enzyme production culture medium, culturing until white hyphae are fully distributed on the surface of the enzyme production culture medium and spores appear, taking out the culture, drying and crushing to obtain the fungal enzyme.
Preferably, in step 2, the sterilization temperature is 121 ℃, and the sterilization time is 60min.
Preferably, in step 3, the temperature of the cultivation is 28-30 ℃.
Preferably, in step 3, the temperature of the drying is 40-45 ℃ and the drying time is 36-72 hours.
Preferably, in step 3, the pulverization is carried out to 20-100 mesh.
(III) a method for using fungal enzyme for increasing the content of gasifiable oil in crude oil, comprising the following steps:
step 1, adding water into fungal enzyme, oscillating by a shaking table, and filtering residues by using glass fibers as a medium to obtain an activated leaching solution of the fungal enzyme preparation;
and step 2, adding the fungal enzyme preparation activated leaching solution into crude oil, and carrying out enzymolysis.
Preferably, in the step 1, the oscillating temperature of the oscillating table is 28-30 ℃, the oscillating speed of the oscillating table is 100-120r/min, and the oscillating time of the oscillating table is 12-18h.
Preferably, in step 2, the concentration of the fungal enzyme preparation is between 14 and 20g/L.
Preferably, in the step 2, the enzymolysis temperature is 37-42 ℃ and the enzymolysis time is 2-5d.
Compared with the prior art, the invention has the beneficial effects that:
the invention identifies 4 strains of crude oil degrading fungi, researches the influence of the enzymatic conversion of 4 strains of fungal enzyme preparations on crude oil group composition and components which can be gasified at 230 ℃, and verifies the enzymolysis of fungal enzyme on asphaltene by using pure asphaltene. The results show that the fungal enzyme preparation is utilized to carry out enzymatic conversion, high polymer components including asphaltene in crude oil can be degraded and converted into micromolecular vaporizable components, and the content of saturated hydrocarbon and aromatic hydrocarbon in enzymatic conversion products of the crude oil is obviously improved.
Drawings
The invention will now be described in further detail with reference to the drawings and to specific examples.
FIG. 1 shows colony morphology of 4 strains of fungi;
FIG. 2 shows the microspore microform characteristics of 4 strains of fungus;
FIG. 3 is a phylogenetic tree of 4 fungi;
FIG. 4 shows the gasifiable components at 230℃after enzymatic conversion and their relative contents, with retention time in min on the abscissa; the ordinate is the absorption value; wherein, the graph (A) is the vaporizable component at 230 ℃ and the relative content of the whole oil converted by an enzyme method; FIG. B shows the 230 ℃ vaporizable component and the relative content of enzymatically converted floating crude oil;
FIG. 5 shows the distribution and morphology of asphaltenes on slides after 4 strains of fungi act on the asphaltene slides; wherein, a, b and c are respectively enlarged by 2, 20 and 40 times, black part is asphaltene and white part is transparent spot without asphaltene.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
1 materials and methods
1.1 materials
(1) Test strain: 4 strains in total, Z3 is Aspergillus oryzae, the accession number on Genbank is KT189153, and the accession number is CCTCC NO: M2016789, and the accession address is university of Chinese Wuhan, which is preserved in China center for type culture collection (China) for 12 months and 30 days in 2016; z05 is Aspergillus spelunceus, and the accession number on Genbank is KT189154; z06 is Aphanocladium aranearum, accession number KT189155 on Genbank; z10 is Aspergillus sydowii and the accession number on Genbank is KT189156. The tested strains are separated and screened by a microbial resource research laboratory of the northwest agriculture and forestry science and technology university, the separation source is China prolonged oil field greasy soil, and the separation culture medium takes crude oil as the only carbon source.
(2) Enzyme-producing medium: according to wheat bran: salt solution: mixing the raw materials thoroughly at the ratio of crude oil=10:8:1, weighing 60g of the mixture in a 600mL tissue culture flask, and sterilizing at 121 ℃ for 60min to obtain an enzyme-producing culture medium; wherein the salt solution comprises 5g KNO 3 、1g KH 2 PO 3 、0.5g MgSO 4 ·7H 2 O, 1000mL water.
(3) Pure asphaltenes: the penetration degree of heavy-glue asphaltene produced by Shaanxi Baoli asphaltene Co., ltd is 91 (0.1 mm), the softening point is 46 ℃, and the ductility at 10 ℃ and 15 ℃ is 45cm and 102cm respectively.
(4) Crude oil: the oil sample of the prolonged oil field in Shanxi province in China has the saturated hydrocarbon content of 218.9g/kg, the aromatic hydrocarbon content of 56.0g/kg, the colloid content of 34.7g/kg, the asphaltene content of 333.3g/kg and the unknown component content of 227.0g/kg; viscosity at 37℃was 160 mPas.
1.2 method
(1) Test fungus identification
Observing colony characteristics, observing cell morphology by using a scanning electron microscope, performing ITS sequence analysis, searching for similarity in a Blast program of an NCBI database according to the obtained sequence, and constructing a phylogenetic tree by adopting a Neighbor-joining method of Mega 5.0 software.
(2) Preparation of fungus enzymolysis liquid
And (3) respectively inoculating 4 strains of oil displacement fungi into an enzyme production culture medium, culturing at 28 ℃ until white hyphae are fully distributed on the surface of the enzyme production culture medium and spores appear, taking out all the cultures, putting the cultures into an oven, drying at 45 ℃ for 72 hours, and crushing to 90 meshes to obtain crude enzyme powder of fungal enzyme.
Weighing 1.50g of crude enzyme powder, adding into a 250mL triangular flask filled with 100mL of pure water, oscillating for 15h at 28 ℃ in a shaking table of 120r/min, and activating the enzyme protein after rehydration; filtering the residue with 2-3g glass fiber as medium to obtain filtrate as active leaching solution of fungal enzyme preparation. The crude enzyme liquid dehydrogenase activity was measured as 63.49-80.38U according to the method of Ma Xin et al (2016).
(3) Influence of enzymatic conversion on crude oil chemical composition
18.00g of crude oil is put into 500mL triangular flasks, 300mL of fungal enzyme preparation activated leaching solution with the concentration of 15.0g/L is added into each flask respectively, enzymolysis is carried out for 5 days at 37 ℃, after the enzymolysis is finished, the triangular flasks are placed into a refrigerator at 4 ℃, the crude oil is cooled and solidified into oil shells, the liquid phase in the triangular flasks is carefully poured out, the liquid phase is washed for 3 times by ice and pure water, the oil shells are dissolved in warm water, and the enzymolysis recovered crude oil is obtained and is collected for standby.
2.000g of crude oil recovered by enzymolysis is weighed, dissolved in 35mL of normal hexane, kept stand for 24h, centrifuged at 3500r/min for 5min, and the precipitate and the organic liquid phase are respectively collected. And drying and weighing the precipitate in a dryer to obtain the asphalt quality. The organic phase was subjected to alumina column chromatography to determine the group composition.
Recovering alumina, absorbent cotton and (NH) in chromatographic column after measurement 4 ) 2 SO 4 And (5) drying and weighing, and calculating the difference between the total mass in the column and the initial mass after the chromatography is finished to obtain the mass of the unknown components remained in the chromatographic column. The above assays were repeated 3 times. Calculating the proportion P of the mass of each component to the total mass of the crude oil according to the formula (1), and calculating the multiplying power n of the content of each component and the content of the corresponding component of the comparison treatment by an enzymatic conversion method according to the formula (2).
The total content of saturated hydrocarbons and aromatic hydrocarbons in the group composition is defined as the vaporizable oil content in the crude oil; the ratio of the content of the gasifiable oil to the sum of the content of the crude oil is the proportion of the gasifiable oil in the crude oil; the difference between the ratio of gasifiable oil in the enzymatic conversion treatment and the ratio of gasifiable oil in the control is defined as the gasifiable oil increase rate delta P in the crude oil, and the calculation formula is shown in formula (3).
In the formula (1): w (W) 2 W and W 1 Respectively represents the total mass and empty bottle mass of the receiving bottle and a certain component during the chromatographic separation of the alumina column, W 0 Indicating the total mass of the sample added to the column. In the formula (2): dt and Dck are the values of the respective parameters measured by the enzymatic conversion treatment and the control. In the formula (3): p (P) a 、P b P Total The sum of the saturated hydrocarbon content and the aromatic hydrocarbon content is shown as a figure, and T, CK shows the enzymatic conversion treatment and the control.
(4) Influence of enzymatic conversion on the vaporizable Components of crude oil at 230 ℃
Converted crude oil a (recovered whole crude oil, abbreviated as "whole oil"): the crude oil recovered by enzymolysis in the above (2) was weighed to 0.200g, dissolved in 3.5mL of n-hexane, and the peak area of each component which can be gasified at 230℃in the crude oil was measured by a gas chromatograph (model Trace GC Ultra, thermo Finnigan Co.). The influence of the crude enzyme solution on the content of each component in crude oil is reflected by the relative content difference of the same component corresponding to the same retention time of the treatment and the control, and the increasing rate of the relative content of each component of the treatment and the control is calculated according to the formula (4).
In the formula (4): st and Sck are peak areas corresponding to the treatments and controls, respectively.
Converted crude oil B (recovered filter paper desorbed crude oil, as it is in a floating state, abbreviated as "floating oil"): cutting rapid qualitative filter paper into 4.5cm×4.5cm filter paper, adsorbing crude oil on the filter paper, placing the filter paper adsorbed with crude oil into 100mL triangular flask, adding 50mL fungal enzyme solution, and performing enzymolysis at 37deg.C for 3d. After the enzymolysis is finished, 10mL of normal hexane solution is added into a triangular flask, crude oil filter paper is dissolved by crude oil which is desorbed after enzyme solution action and floats on the upper layer of the liquid surface, the upper normal hexane phase is carefully sucked out, and the relative content (expressed by peak area) of the vaporizable component at 230 ℃ in the floating crude oil is measured by a gas chromatograph.
(5) Influence of enzymatic conversion on pure asphaltene Components
Preparation of an asphaltene solution: 5 drops of asphaltene solution dissolved in carbon tetrachloride were added to the weighed (m 0 ) On the slide, the slide is rotated to spread the asphaltene solution, and after the carbon tetrachloride is naturally volatilized, the asphaltene is uniformly attached to the slide and then weighed (m 1 ). After the asphaltene glass slide was irradiated with ultraviolet light for 30 minutes, the asphaltene glass slide was horizontally placed in a 500mL wide-mouth glass bottle containing 100mL of crude enzyme solution, and subjected to enzymolysis at 37℃for 35d, and gently shaken 4 times per day. After the enzymolysis is finished, taking out the asphaltene glass slide, gently flushing with distilled water for 3 times, naturally air-drying, and weighing the mass (m 2 ) The method comprises the steps of carrying out a first treatment on the surface of the 100mL of carbon tetrachloride was added to the crude enzyme solution, the mixture was shaken well, the organic phase was recovered, and the mixture was evaporated to dryness and weighed (m 3 ). Crude enzyme liquid of fungi to asphaltThe degradation amount of the mass is m 1 +m 3 -m 2 The ratio of the amount of asphaltene degradation by the crude enzyme solution to the amount of initial asphaltene attachment was defined as asphaltene degradation rate (Asphalt Degradation Efficiency, ADE%) and calculated according to equation (5). The ratio n of the content of the certain component of the enzyme treatment to the content of the certain component of the control is calculated as the enzyme treatment multiplying power by the formula (2). The micro-morphology observation method after the degradation of the pure asphaltene is shown in Gao (2017 a), and the method for measuring the influence on the components refers to the method for measuring the influence on the chemical components of the crude oil by the enzymatic conversion.
(6) Result calculation and data processing
All data were subjected to correlation analysis and difference significance testing using SAS 9.2 (SAS Institute Inc, cary, NC, USA).
2. Test results and analysis
(1) Fungus identification
Colony morphological characteristics and thallus microscopic morphology observation: the colony morphology and spore-forming structure microscopic morphology of the 4 strains of fungi are shown in fig. 1 and 2, respectively. By microscopic morphological observation of colony morphology features and spore-forming structures of the strain, it was initially identified that Z3, Z05 and Z10 are all Aspergillus sp according to morphological descriptions of the Chinese fungus (Ji Zu and 1997).
The fungal ITS sequence was determined as follows:
z3Aspergillus oryzae strain with accession number KT189153 and sequence shown as SEQ ID No.1, specifically comprises the following steps:
GCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCG GCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTT GATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCA GAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGG CTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCATCCTCGAGCGTATGGGGCTTTGTCACCC GCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATC AAGCCGGAGGAAA。
z05Aspergillus spelunceus strain with the accession number KT189154 and the sequence shown as SEQ ID No.2 is as follows:
TGACTACCTAACACTGTTGCTTCGGCGGGGAGCGCCCCTCGGGGA GCGAGCCGCCGGGGACCACCGAACTTCATGCCTGAGAGTAATGCAGTCTGAGCCTGAATAGTATAATCAGTCAAAACTTTCAACAATGGATCTCTTG GTTCCGGCATCGATGAAGAACGCAGCGAACTGCGATAAGTAATGTGAA TTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAG CCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCTTCCGGGGAGGGACGGACCCGAAAGGCAGTGGCGGCACCGTGTCCGGTCCTCGAGCGTATGGGGCT TTGTCACCCGCTCGACTAGGGCCGGCCGGGCGCCAGCCGGCGTCTCCA ACCATTTTTTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGA ACTTAAGCATATCAATAAGCGGAG。
z06Aphanocladium aranearum strain with accession number KT189155 and sequence shown as SEQ ID No.3, specifically as follows:
TGAACATACCACGATGTTGCTTCGGCGGACTCGCCCCGGCGTCCGG ACGGCCTAGCGCCGCCCGCGGCCCGGATCCAGGCGGCCGCCGGAGACC ACCAAAACTATTTTGTATCAGCAGTTTTTTCTGAATCCGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATC GATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGG CGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACTTCCCTTTGGGG AAATCGGCGTTGGGGACTGGCAGCATACCGCCGGCCCCGAAATGGAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTAATCCAACCTCGCACCG GAACCCCGACGTGGCCACGCCGTAAAACACCCCACTTTCTGAACGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAG CGGA。
z10Aspergillus sydowii strain with accession number KT189156 and sequence shown as SEQ ID No.4, specifically as follows:
CCTCCGGGCGCCCAACCTCCCACCCGTGAATACCTAACACTGTTGC TTCGGCGGGGAACCCCCTCGGGGGCGAGCCGCCGGGGACTACTGAACTTCATGCCTGAGAGTGATGCAGTCTGAGTCTGAATATAAAATCAGTCAA AACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGC GAACTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCC GAGCGTCATTGCTGCCCATCAAGCCCGGCTTGTGTGTTGGGTCGTCGTCCCCCCCGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGTGTCCGGTC CTCGAGCGTATGGGGCTTTGTCACCCGCTCGACTAGGGCCGGCCGGGCGCCAGCCGACGTCTCCAACCATTTTTCTTCAGGTTGACCTCGGATCAGG TAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA。
based on ITS sequence determination, 4 phylogenetic trees of fungi were constructed using MEGA4.0 software (fig. 3), with Z3 determined to be Aspergillus oryzae, Z05Aspergillus spelunceus, Z06Aphanocladium aranearum, and Z10Aspergillus sydowii.
(2) Enzymatic conversion products of crude oil by fungal enzymes
2.1 saturated hydrocarbons and aromatic hydrocarbons
Saturated hydrocarbons and aromatic hydrocarbons are the main components of gasoline, kerosene and diesel oil. As shown in Table 1, the saturated hydrocarbon, aromatic hydrocarbon and gum contents in the crude oil after enzymatic conversion are greatly increased by 2.2-2.5 times, 1.3-2.1 times and 1.2-1.8 times respectively compared with the control, wherein the saturated hydrocarbon and aromatic hydrocarbon contents reach remarkable levels (P < 0.05) compared with the amplification of the control. The increase rate of the gasifiable oil (the total content of saturated hydrocarbon and aromatic hydrocarbon) caused by enzymatic conversion is 30.3% -44.4% compared with the control, wherein the gasifiable oil content in crude oil is respectively increased by 44.4% and 42.1% through enzymatic conversion of fungal enzyme preparations Z3 and Z10. During enzymatic conversion, the asphaltene content and the unknown components and other heavy components are significantly reduced (P < 0.05), the asphaltene content in the conversion product is only 1/5-1/2 of that of the control, and the unknown components are about 1/2 of that of the control.
As seen in Table 1, the enzyme dehydrogenase activity of the 4 fungal enzyme preparations is 63.49U-80.38U; the dehydrogenase activity is significantly positively correlated (P < 0.01) with the saturated hydrocarbon content in the enzymatic conversion product, and significantly (P < 0.05) or very significantly (P < 0.01) with the bitumen and unknown components, indicating that the saturated hydrocarbon fraction of the enzymatic conversion increase is derived from the degradation of asphaltenes and unknown components in the crude oil. The increase rate of the gasifiable oil treated corresponding to Z3 and Z10 with higher dehydrogenase activity is also higher, and the causal relationship between the increase of gasifiable oil in the enzymatic conversion product and the dehydrogenase activity is also disclosed. However, the correlation of dehydrogenase activity with the increase rate of gasifiable oil did not reach statistically significant levels (P > 0.05).
TABLE 1 fungal enzyme dehydrogenase Activity and content of 4 Main Components in crude oil after enzymatic conversion
In table 1: the different lower case letters after the same column of data represent significant differences between treatments (P < 0.05); r is the correlation coefficient of the content of each component in crude oil and the dehydrogenase activity of the corresponding crude enzyme preparation, and represents obvious correlation (P < 0.05) and extremely obvious correlation (P < 0.01) respectively.
2.2 Component capable of gasifying at 230 DEG C
As is clear from Table 2 and FIG. 4A, in the enzymatic conversion products of the crude oil (whole oil) to be tested, 23 gasification components at 230℃were detected in total. In the crude oil converted by the enzymatic method of the fungal enzyme preparations Z05 and Z06, the relative contents of 22 components are obviously increased compared with the comparison, and the average increases are 59.1 percent and 76.1 percent respectively, which indicates that the content of gasifiable oil in the crude oil is greatly increased by the enzymatic method conversion, and the heavy components in the crude oil are converted into light components which can be gasified at 230 ℃. The conversion effect of the other two fungal enzyme preparations is slightly poorer.
TABLE 2 relative content of components gasifiable at 230℃in enzymatically converted crude oil (whole oil)
As can be seen from FIG. 4B and Table 3, 14 components which can be gasified at 230℃were detected in total from the crude oil in a floating state (filter paper desorbed crude oil) obtained by the enzymatic conversion. Wherein, in the floating crude oil converted by the fungal enzymes Z3 and Z06, the relative contents of components 1 to 11 with retention time of 13 to 31min are respectively increased by 19.8 to 97.4 percent and 34.3 to 113.8 percent compared with the control, which indicates that the contents of light components in most of the components which can be gasified at 230 ℃ in the crude oil are increased, namely the content of the gasifiable oil in the crude oil is increased through the enzymatic conversion. At the same time, the relative content of components 12-14 with retention time of 32-35min is reduced compared with control. The relative contents of components 1 to 7 and 1 to 10 obtained by the enzymatic conversion of fungi Z05 and Z10 are respectively increased by 8.9 to 89.7 percent and 2.5 to 82.9 percent compared with the comparison, the relative contents of the other components are slightly reduced, and the enzymatic conversion effect on crude oil is poor.
TABLE 3 relative content of vaporizable Components at 230℃in enzymatically converted crude oil (Floating oil)
2.3 enzymatic conversion products of pure asphaltenes
As can be seen from Table 4, the degradation rate of 4 strains of fungal enzymes on pure asphaltenes on asphaltene slides was 9.1% -14.2% by fungal enzyme treatment, 39.7-61.6 times that of the control, and the difference from the control reached a significant level (P < 0.05). Wherein the fungal enzymes corresponding to Z3 and Z10 have the best degradation effect on pure asphaltenes. The saturated hydrocarbon content in the pure asphaltene is 1.0-1.2 times of that of the control by the fungal enzyme solution treatment; the contents of aromatic hydrocarbon, colloid and unknown components are respectively 0.9-1.0 times, 0.2-0.6 times (P < 0.05) and 0.4-0.5 times (P < 0.05) of the control. The increase rate of the gasifiable oil is 13.2% -17.5%, wherein the fungal enzymes corresponding to Z3 and Z10 can increase the gasifiable oil content in the pure asphaltene by 17.4% -17.5%.
As can be seen from fig. 5, the micro morphology of the slide attached to asphaltenes was significantly changed by the fungal enzyme treatment. When the glass slide is amplified by 2 times, pure asphaltene attached to the surface of the control glass slide is uniformly opaque black, and more transparent spots appear on the surface of the asphaltene attached to the glass slide after enzyme liquid treatment, wherein the transparent spots on the asphaltene attached to the glass slide after Z3 treatment are more. At 20-fold magnification, the control had only a small amount of small plaque, while the enzyme solution treated slide had much more of the plaque area on the attached asphaltenes than the control. When the magnification is 40 times, asphaltenes attached to the control treatment glass slide are in a thin and uniform state, and asphaltenes attached to the enzyme solution treatment glass slide are in aggregation bulge states with different degrees.
TABLE 4 degradation rate of asphaltene after enzymatic conversion and composition of asphaltene enzymolysis product family
The invention identifies 4 strains of crude oil degrading fungi, researches the influence of the enzymatic conversion of 4 strains of fungal enzyme preparations on crude oil group composition and components which can be gasified at 230 ℃, and verifies the enzymolysis of fungal enzyme on asphaltene by using pure asphaltene. The results show that the fungal enzyme preparation is utilized to carry out enzymatic conversion, high polymer components including asphaltene in crude oil can be degraded and converted into micromolecular vaporizable components, and the content of saturated hydrocarbon and aromatic hydrocarbon in enzymatic conversion products of the crude oil is obviously improved.
According to the components and structural characteristics of the compounds in crude oil, the compounds are divided into saturated hydrocarbon, aromatic hydrocarbon, colloid, asphaltene and other components. Gum and asphaltenes are the largest molecular weight components of crude oil. Asphaltene has a complex structure, high content in thick oil, and great influence on the viscosity of crude oil, and influences the exploitation and utilization of crude oil. Recent studies have found that pure asphaltenes can be degraded by Pseudomonas aeruginosa (Pseudomonas aeruginosa) with degradation rates up to 10%, but there are few studies on the degradation of asphaltenes by fungal enzyme preparations.
The research shows that the fungal enzyme preparation has a strong degradation effect on asphaltenes in crude oil, and the degradation rate of fungal enzyme Z10 on asphaltenes in crude oil is up to 86.4%. The fungal enzyme also has good degradation effect on the pure asphaltene, can increase the saturated hydrocarbon content in the degradation product of the pure asphaltene, reduce the content of aromatic hydrocarbon, colloid and unknown components, and change the asphaltene from a uniform adhesion state to a raised aggregation state, which is related to degradation of long-chain components in the asphaltene into short-chain alkane, reduction of viscosity and enhancement of fluidity, and the result proves that the degradation effect of the fungal enzyme preparation on the asphaltene in crude oil does exist. The strong degradation of asphaltene by fungal enzyme preparations is one of the mechanisms for increasing the content of gasifiable oil in crude oil converted by enzymatic methods. The primary enzyme of enzymatic conversion is dehydrogenase. When crude oil is converted by a dehydrogenase enzymatic method, the dehydrogenase activity significantly affects the contents of saturated hydrocarbons (light components) and asphaltenes and unknown components (heavy components). In the present invention, there is a significant or very significant correlation between dehydrogenase activity and saturated hydrocarbon and asphaltene and unknown component content, revealing the mechanism by which enzymatic conversion leads to a substantial increase in vaporizable hydrocarbons: enzymatic degradation of heavy components.
In conclusion, the invention can degrade and convert heavy macromolecular components such as asphaltene in crude oil into micromolecular vaporizable components through enzymatic conversion of the fungal enzyme preparation, so that the content of vaporizable components in the crude oil is greatly improved, the quality of the crude oil is improved, the yield of vaporizable oils such as gasoline, low-boiling kerosene and diesel oil during refining of the crude oil is improved, theoretical support is provided for improving the quality of the crude oil by utilizing the fungal enzyme, and scientific basis and a feasible technical scheme are provided for technological design of improving the yield of gasoline and low-boiling coal and diesel oil during refining of the crude oil through enzymatic conversion of the fungal enzyme.
While the invention has been described in detail in this specification with reference to the general description and the specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Shaanxi Boqin bioengineering Limited northwest university of agriculture and forestry science and technology
<120> fungal enzyme for increasing the content of gasifiable oil in crude oil, and method for preparing and using the same
<130> 2018
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 546
<212> DNA
<213> Aspergillus oryzae strain
<400> 1
gcgagcccaa cctcccaccc gtgtttactg taccttagtt gcttcggcgg gcccgccatt 60
catggccgcc gggggctctc agccccgggc ccgcgcccgc cggagacacc acgaactctg 120
tctgatctag tgaagtctga gttgattgta tcgcaatcag ttaaaacttt caacaatgga 180
tctcttggtt ccggcatcga tgaagaacgc agcgaaatgc gataactagt gtgaattgca 240
gaattccgtg aatcatcgag tctttgaacg cacattgcgc cccctggtat tccggggggc 300
atgcctgtcc gagcgtcatt gctgcccatc aagcacggct tgtgtgttgg gtcgtcgtcc 360
cctctccggg ggggacgggc cccaaaggca gcggcggcac cgcgtccatc ctcgagcgta 420
tggggctttg tcacccgctc tgtaggcccg gccggcgctt gccgaacgca aatcaatctt 480
tttccaggtt gacctcggat caggtaggga tacccgctga acttaagcat atcaagccgg 540
aggaaa 546
<210> 2
<211> 506
<212> DNA
<213> Aspergillus spelunceus strain
<400> 2
tgactaccta acactgttgc ttcggcgggg agcgcccctc ggggagcgag ccgccgggga 60
ccaccgaact tcatgcctga gagtaatgca gtctgagcct gaatagtata atcagtcaaa 120
actttcaaca atggatctct tggttccggc atcgatgaag aacgcagcga actgcgataa 180
gtaatgtgaa ttgcagaatt cagtgaatca tcgagtcttt gaacgcacat tgcgccccct 240
ggcattccgg ggggcatgcc tgtccgagcg tcattgctgc ccatcaagcc cggcttgtgt 300
gttgggtcgt cgtcccccct tccggggagg gacggacccg aaaggcagtg gcggcaccgt 360
gtccggtcct cgagcgtatg gggctttgtc acccgctcga ctagggccgg ccgggcgcca 420
gccggcgtct ccaaccattt ttttttcagg ttgacctcgg atcaggtagg gatacccgct 480
gaacttaagc atatcaataa gcggag 506
<210> 3
<211> 534
<212> DNA
<213> Aphanocladium aranearum strain
<400> 3
tgaacatacc acgatgttgc ttcggcggac tcgccccggc gtccggacgg cctagcgccg 60
cccgcggccc ggatccaggc ggccgccgga gaccaccaaa actattttgt atcagcagtt 120
ttttctgaat ccgccgcaag gcaaaacaaa tgaatcaaaa ctttcaacaa cggatctctt 180
ggttctggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc 240
agtgaatcat cgaatctttg aacgcacatt gcgcccgcca gcattctggc gggcatgcct 300
gttcgagcgt catttcaacc ctcgacttcc ctttggggaa atcggcgttg gggactggca 360
gcataccgcc ggccccgaaa tggagtggcg gcccgtccgc ggcgacctct gcgtagtaat 420
ccaacctcgc accggaaccc cgacgtggcc acgccgtaaa acaccccact ttctgaacgt 480
tgacctcgga tcaggtagga atacccgctg aacttaagca tatcaataag cgga 534
<210> 4
<211> 525
<212> DNA
<213> Aspergillus sydowii strain
<400> 4
cctccgggcg cccaacctcc cacccgtgaa tacctaacac tgttgcttcg gcggggaacc 60
ccctcggggg cgagccgccg gggactactg aacttcatgc ctgagagtga tgcagtctga 120
gtctgaatat aaaatcagtc aaaactttca acaatggatc tcttggttcc ggcatcgatg 180
aagaacgcag cgaactgcga taagtaatgt gaattgcaga attcagtgaa tcatcgagtc 240
tttgaacgca cattgcgccc cctggcattc cggggggcat gcctgtccga gcgtcattgc 300
tgcccatcaa gcccggcttg tgtgttgggt cgtcgtcccc cccgggggac gggcccgaaa 360
ggcagcggcg gcaccgtgtc cggtcctcga gcgtatgggg ctttgtcacc cgctcgacta 420
gggccggccg ggcgccagcc gacgtctcca accatttttc ttcaggttga cctcggatca 480
ggtagggata cccgctgaac ttaagcatat caataagcgg aggaa 525
Claims (3)
1. Use of a fungal enzyme for increasing the content of gasifiable oil in crude oil, wherein the preparation of the fungal enzyme comprises the steps of:
step 1, mixing potassium nitrate, monopotassium phosphate, anhydrous magnesium sulfate and water to obtain a salt solution;
step 2, uniformly mixing the salt solution with wheat bran and crude oil, and sterilizing to obtain an enzyme-producing culture medium;
step 3, inoculating the oil displacement fungi into the enzyme production culture medium, culturing until white hyphae are fully distributed on the surface of the enzyme production culture medium and spores appear, taking out the culture, drying and crushing to obtain the fungal enzyme;
the oil displacement fungi isAspergillus oryzae Z3、Aspergillus spelunceus Z05、 Aphanocladium aranearumZ06 orAspergillus sydowii Z10；
In the step 3, the temperature of the culture is 28-30 ℃; the temperature of the drying is 40-45 ℃, and the drying time is 36-72 hours.
2. Use according to claim 1, wherein the fungal enzyme comprises the following raw materials: an oil displacement fungus and an enzyme production culture medium; wherein the enzyme-producing medium comprises wheat bran, salt solution and crude oil;
the oil displacement fungi isAspergillus oryzae Z3、Aspergillus spelunceus Z05、 Aphanocladium aranearum Z06. Or (b)Aspergillus sydowii Z10；
The inoculation amount of the oil displacement fungi is 0.1% -0.5%;
the salt solution comprises potassium nitrate, monopotassium phosphate, anhydrous magnesium sulfate and water;
the enzyme-producing culture medium comprises 7-13 parts of wheat bran, 5-11 parts of salt solution and 0.5-2.0 parts of crude oil; wherein the salt solution comprises 2-8 parts of potassium nitrate, 0.2-2 parts of monopotassium phosphate, 0.1-0.9 part of anhydrous magnesium sulfate and 1000 parts of water.
3. Use according to claim 1, wherein the use of the fungal enzyme comprises the steps of:
step 1, adding water into fungal enzyme, oscillating by a shaking table, and filtering residues by using glass fibers as a medium to obtain an activated leaching solution of the fungal enzyme preparation;
step 2, adding the fungal enzyme preparation activated leaching solution into crude oil for enzymolysis;
in the step 1, the oscillating temperature of the shaking table is 28-30 ℃, the oscillating rotating speed of the shaking table is 100-120r/min, and the oscillating time of the shaking table is 12-18h;
in the step 2, the concentration of the fungal enzyme preparation is 14-20g/L; the enzymolysis temperature is 37-42 ℃, and the enzymolysis time is 2-5d.
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| CN109486775A (en) | 2019-03-19 |
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