CN113528363A - Recombinant strain, composite strain and petroleum hydrocarbon biodegradation method - Google Patents

Recombinant strain, composite strain and petroleum hydrocarbon biodegradation method Download PDF

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CN113528363A
CN113528363A CN202110499958.XA CN202110499958A CN113528363A CN 113528363 A CN113528363 A CN 113528363A CN 202110499958 A CN202110499958 A CN 202110499958A CN 113528363 A CN113528363 A CN 113528363A
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degradation
gene
hexadecane
petroleum hydrocarbon
strain
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CN113528363B (en
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元英进
李恒昌
丁明珠
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Tianjin University
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Abstract

The invention relates to the technical field of bioremediation, in particular to a recombinant strain, a composite strain and a petroleum hydrocarbon biodegradation method. The invention adopts yeast as the chassis cell for research and constructs the recombinant strain capable of degrading petroleum hydrocarbon. Based on the characteristics of clear genetic background of yeast and convenient molecular operation, the recombinant strain is simpler and easier to construct and obtain. In the invention, after genes for degrading alkane are introduced into saccharomyces cerevisiae and successfully expressed, the research on petroleum hydrocarbon degradation is carried out by using strains with higher degradation rate, and the degradation conditions are further optimized, so that the degradation efficiency can finally reach 91.63%.

Description

Recombinant strain, composite strain and petroleum hydrocarbon biodegradation method
Technical Field
The invention relates to the technical field of bioremediation, in particular to a recombinant strain, a composite strain and a petroleum hydrocarbon biodegradation method.
Background
Petroleum hydrocarbons, including gasoline, kerosene, diesel oil, lubricating oil, paraffin, and asphalt, are mixtures of various hydrocarbons (n-paraffins, branched paraffins, naphthenes, aromatics) and small amounts of other organic compounds, such as sulfides, nitrides, naphthenic acids, and the like. With the development of economy, the demand of human beings for energy is continuously expanding, and petroleum becomes one of the most important energy sources of human beings. However, during the process of oil extraction, processing and utilization, more and more oil may enter the soil environment and the ocean, thereby causing pollution and destruction of the soil environment and the ocean water quality. Once in the soil, the excessive total petroleum hydrocarbon is difficult to remove, and causes serious harm to the society, the economy and the human beings. And excessive petroleum hydrocarbon enters the ocean, can be gathered in marine organisms and enter human bodies along with food chains, thus being harmful to human health.
Bioremediation is a main restoration mode which accords with the concept of green development and gradually becomes the key point of research. Bioremediation of petroleum hydrocarbon-contaminated environments can be roughly divided into three processes, namely an interphase adaptation process, a transport process and a degradation process of microorganisms to petroleum hydrocarbons. In the process of bioremediation of petroleum hydrocarbon polluted environment, microorganisms firstly carry out an interphase adaptation process on the petroleum hydrocarbon, and in the process, the microorganisms generally increase the bioavailability of the petroleum hydrocarbon pollutants by two modes of surfactant secretion and chemotactic movement; then, petroleum hydrocarbon enters cells through a transfer process, the main modes include free diffusion, passive transport, active transport and endocytosis, and most of the petroleum hydrocarbon transfer process needs the participation of transfer protein; finally, the petroleum hydrocarbon is degraded in the cell, once the petroleum hydrocarbon molecules enter the cell, the petroleum hydrocarbon molecules are oxidized into aliphatic alcohol by oxygenase, the aliphatic alcohol is sequentially converted into aliphatic acid, fatty acyl coenzyme A and the like, and then the degradation is carried out through beta-oxidation.
The existing research on petroleum hydrocarbon degrading bacteria focuses on the field of bacteria, but the research on a mechanism for degrading petroleum hydrocarbon is not enough, and a strain with clear genetic background is selected as a chassis cell, so that the molecular operation is convenient, the degradation mechanism of the petroleum hydrocarbon can be better proved, and the degradation rate of the bacteria on the petroleum hydrocarbon can be further improved.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a recombinant yeast strain capable of degrading petroleum hydrocarbon, a composite strain capable of degrading petroleum hydrocarbon, and a method for improving the biodegradation of petroleum hydrocarbon.
The recombinant yeast provided by the invention expresses alkane degrading genes;
the alkane degrading genes include: SYHY gene, AlmA gene or almm gene.
The invention provides a recombinant yeast expressing SYHY gene. In some embodiments, the nucleic acid sequence of the SYHY gene is shown as SEQ ID NO 1.
The invention also provides a recombinant yeast expressing the AlmA gene. In some embodiments, the nucleic acid sequence of the AlmA gene is shown in SEQ ID NO 2.
The invention also provides a recombinant yeast for expressing the alk gene. In some embodiments, the nucleic acid sequence of the alk gene is set forth in SEQ ID NO 3.
The chassis bacteria of the recombinant yeast are saccharomyces cerevisiae or synthetic yeast only containing X chromosome.
In some embodiments, the substrate bacterium is saccharomyces cerevisiae BY 4741.
Research shows that the saccharomyces cerevisiae BY4741 expressing the AlmA gene of the nucleic acid sequence shown as SEQ ID NO. 2 has higher degradation efficiency on n-hexadecane.
The construction method of the recombinant yeast comprises the following steps: the alkane degrading gene is introduced into yeast.
The introduction of the present invention includes constructing plasmid vector containing alkane degrading gene and converting into saccharomycete. The plasmid vector is an integrative plasmid vector or an episomal plasmid vector. In the present embodiment, the plasmid vector is an integrative plasmid vector. In some embodiments, the integrative plasmid is pRS416 and the insertion site of the alkane-degrading gene on the plasmid vector is between Hind III and BamH I.
The recombinant yeast is applied to degrading petroleum hydrocarbon.
The invention provides a bacterial agent A for degrading petroleum hydrocarbon, which comprises the recombinant yeast.
The microbial inoculum A also comprises a culture medium and/or a surfactant; the culture medium is an SC culture medium; the surfactant is at least one of rhamnolipid, sophorolipid and tween.
The invention also provides a bacterial agent B for degrading petroleum hydrocarbon, which comprises the recombinant yeast and pseudomonas aeruginosa.
In the microbial inoculum B, the quantity ratio of the recombinant yeast to the pseudomonas aeruginosa is (1-4): (1-4).
The pseudomonas aeruginosa is a species that can convert petroleum hydrocarbons to surfactants. The pseudomonas aeruginosa is 1A00364, 1A01151 or 1A 06466.
The microbial inoculum also comprises an LB culture medium. The concentration of NaCl in the LB culture medium is 10g/L, and the pH value is 7.5.
The invention also provides a method for degrading petroleum hydrocarbon, which uses the bacterial agent A or B to degrade the petroleum hydrocarbon. In some embodiments, the petroleum hydrocarbon comprises n-hexadecane.
Degrading by using the microbial inoculum A:
in the degradation system, the initial OD of the microbial inoculum A600The value is 0.2, the concentration of the petroleum hydrocarbon is 4g/L, and the concentration of the surface active agent is 300 mg/L-4 g/L;
the degradation conditions comprise that the temperature is 30 ℃, and the vibration degradation is carried out at 220rpm for 3-4 days.
Degrading by using the microbial inoculum B:
in the degradation system, the initial OD of the microbial inoculum B600The value is 0.2, and the concentration of the petroleum hydrocarbon is 4 g/L;
the degradation conditions comprise that the temperature is 30 ℃, and the vibration degradation is carried out at 220rpm for 3-4 days.
The invention adopts yeast as the chassis cell for research and constructs the recombinant strain capable of degrading petroleum hydrocarbon. Based on the characteristics of clear genetic background of yeast and convenient molecular operation, the recombinant strain is simpler and easier to construct and obtain. In the invention, after genes for degrading alkane are introduced into saccharomyces cerevisiae and successfully expressed, the research on petroleum hydrocarbon degradation is carried out by using strains with higher degradation rate, and the degradation conditions are further optimized, so that the degradation efficiency can finally reach 91.63%.
Drawings
FIG. 1 shows the case where Saccharomyces cerevisiae BY4741 utilizes n-hexadecane, wherein 1-a shows that Saccharomyces cerevisiae BY474 cannot utilize n-hexadecane for solid plate validation, and 1-b shows that Saccharomyces cerevisiae BY4741 cannot utilize n-hexadecane for liquid fermentation validation;
FIG. 2 shows the principle of Saccharomyces cerevisiae degradation of n-hexadecane;
FIG. 3 shows BY4741, SYHY, AlmA, and almm using n-hexadecane, where a shows the growth curve; b represents the degradation rate of 96 h;
FIG. 4 shows the growth and degradation of n-hexadecane by strains screened using the genomic rearrangement technique, wherein a shows the growth curves of SYHYX-0 to SYHYX-8; b represents the 96h degradation rate of SYHYX-0 to SYHYX-8; c shows the growth curves of AlmAX-0 to AlmAX-16; d represents the 96h degradation rate of AlmAX-0 to AlmAX-16; e shows the growth curves from alkmX-0 to alkmX-11; f represents the degradation rate of alkmX-0 to alkmX-11 for 96 h;
FIG. 5 shows the case of using n-hexadecane when a single surfactant is added, wherein a shows a growth curve; b represents the degradation rate of 96h, in the figure, T represents Tween 40, R represents rhamnolipid, and S represents sophorolipid;
FIG. 6 shows the effect of different concentration gradients of surfactants on the degradation of n-hexadecane, wherein a shows the growth curve of different sophorolipid concentrations, b shows the degradation rate of 96h at different sophorolipid concentrations, c shows the growth curve of different rhamnolipid concentrations, and d shows the degradation rate of 96h at different sophorolipid concentrations; in the figure, R represents rhamnolipid, S represents sophorolipid;
fig. 7 shows the case of using n-hexadecane when two surfactants were added: wherein a represents each group of growth curves, b represents the degradation rate of 96h of n-hexadecane, T represents Tween 40, R represents rhamnolipid, and S represents sophorolipid;
fig. 8 shows the case of using n-hexadecane when three surfactants were added: wherein a represents each group of growth curves, b represents the degradation rate of 96h of n-hexadecane, T represents Tween 40, R represents rhamnolipid, and S represents sophorolipid;
fig. 9 shows the case of exogenous addition of different combinations of surfactants using n-hexadecane: wherein a represents each group of growth curves, b represents the degradation rate of 96h of n-hexadecane, T represents Tween 40, R represents rhamnolipid, and S represents sophorolipid;
FIG. 10 shows the degradation of n-hexadecane by three P.aeruginosa strains (1A00364, 1A01151, 1A06466) for 48h, wherein a shows the growth curves of each group and b shows the degradation rate of n-hexadecane for 48 h;
FIG. 11 shows the case where the engineered strain AlmA shares, respectively, n-hexadecane with three Pseudomonas aeruginosa strains (1A00364, 1A01151, 1A 06466);
FIG. 12 shows the effect of optimization of fermentation conditions on degradation efficiency, wherein a shows the effect of inoculation ratio on degradation efficiency; b shows the effect of temperature on degradation efficiency; c shows the effect of pH on degradation efficiency; d shows the influence of NaCl concentration on degradation efficiency;
FIG. 13 shows a comparison of the degradation rates of n-hexadecane before and after optimization of fermentation conditions.
Detailed Description
The invention provides a recombinant strain, a composite strain and a petroleum hydrocarbon biodegradation method, and a person skilled in the art can use the contents to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The recombinant yeast of the present invention refers to a yeast strain capable of expressing one or more heterologous nucleic acid sequences. The heterologous nucleic acid expressed in the recombinant yeast of the present invention is an alkane-degrading gene. The alkane degrading enzyme gene comprises at least one of SYHY gene, AlmA gene or alk m gene. In embodiments of the invention, a heterologous nucleic acid is expressed in the recombinant yeast. Namely, in the recombinant yeast of the present invention, any one of SYHY gene, AlmA gene or almm gene is expressed. The invention provides a recombinant yeast expressing SYHY gene. In some embodiments, the nucleic acid sequence of the SYHY gene is shown as SEQ ID NO 1. The invention also provides a recombinant yeast expressing the AlmA gene. In some embodiments, the nucleic acid sequence of the AlmA gene is shown in SEQ ID NO 2. The invention also provides a recombinant yeast for expressing the alk gene. In some embodiments, the nucleic acid sequence of the alk gene is set forth in SEQ ID NO 3.
The recombinant yeast is constructed by introducing exogenous nucleic acid into a chassis bacterium. The chassis bacteria of the recombinant yeast are saccharomyces cerevisiae or synthetic yeast only containing X chromosome. In some embodiments, the substrate bacterium is saccharomyces cerevisiae BY 4741.
The strain constructed by the invention comprises the following components:
BY4741 Strain transformed with SYHY gene;
BY4741 strain transformed with AlmA gene;
BY4741 strain transformed with the alkm gene;
synthetic yeast transformed with SYHY gene;
synthetic yeast transformed with AlmA gene;
synthetic yeast transformed with the alkm gene.
The experiment of the invention shows that the BY4741 strain for transforming the AlmA gene has higher degradation efficiency on the n-hexadecane, and the degradation rate can reach 14.48 percent in 96 hours.
The construction method of the recombinant yeast strain comprises the step of introducing alkane degradation genes into yeast. The method for introducing a heterologous gene into yeast of the present invention may employ any method known in the art. For example, electric pulse transformation or crisper-mediated gene site-directed integration methods. In the examples of the present invention, the transformation was carried out by electrical transformation. Before transformation, recombinant plasmids are constructed.
The recombinant plasmid comprises an alkane degrading gene, and a skeleton vector of the recombinant plasmid is an integrated plasmid vector or an episomal plasmid vector. For faster introduction of foreign genes, the integrative plasmid in the examples of the present invention was pRS 416. The insertion site of the alkane degrading gene on the plasmid vector is between Hind III and BamH I. The recombinant plasmid constructed and obtained by the invention comprises pRS416-SYHY, pRS416-AlmA and pRS 416-almm.
The recombinant yeast of the invention can degrade petroleum hydrocarbon. The petroleum hydrocarbon comprises normal paraffin, branched paraffin, cyclane and aromatic hydrocarbon. The invention takes the n-hexadecane as an experimental object to identify the degradation effect of the bacterial strain on the petroleum hydrocarbon. The recombinant yeast can independently prepare a microbial inoculum which is used as the only strain to degrade petroleum hydrocarbon; the composite microbial inoculum can also be prepared by compounding with other strains and used for degrading petroleum hydrocarbon.
The microbial inoculum provided by the invention taking the recombinant yeast strain as a petroleum hydrocarbon degrading strain is marked as microbial inoculum A, and the microbial inoculum A can only contain the recombinant yeast and also can comprise a culture medium. The recombinant yeast can be bacterial powder or fresh bacterial liquid. The culture medium is a liquid culture medium or a solid culture medium. The culture medium and the recombinant yeast may be present in a mixture or may be present independently of each other. The culture medium is preferably an SC medium. The medium may also include a surfactant. The surfactant is at least one of rhamnolipid, sophorolipid and tween. In the examples of the present invention, the surfactants attempted to be used include: rhamnolipid, sophorolipid, tween-40, rhamnolipid and sophorolipid, sophorolipid and tween-40, rhamnolipid and tween-40. The result shows that the addition of sophorolipid and tween-40 as surfactants is more beneficial to the degradation of petroleum hydrocarbon by the recombinant yeast.
The recombinant yeast strain and the pseudomonas aeruginosa are used together as a bacterial agent for degrading petroleum hydrocarbon strains and are marked as a bacterial agent B, and the bacterial agent B comprises the recombinant yeast and the pseudomonas aeruginosa. The pseudomonas aeruginosa is a species that can convert petroleum hydrocarbons to surfactants. In some embodiments, the pseudomonas aeruginosa is 1a00364, 1a01151, or 1a 06466. The microbial inoculum B also can comprise a culture medium. The recombinant yeast or the pseudomonas aeruginosa can be bacterial powder or fresh bacterial liquid. In the microbial inoculum B, the quantity ratio of the recombinant yeast to the pseudomonas aeruginosa is (1-4): (1-4). In specific embodiments, the number ratio of the recombinant yeast to the pseudomonas aeruginosa is 4:1, 2:1, 1:2 or 1: 4. The culture medium is a liquid culture medium or a solid culture medium. The culture medium and the recombinant yeast may be present in a mixture or may be present independently of each other. The culture medium is preferably SC-glucose +10 g/LNaCl. The pH was 7.5.
In the method for degrading petroleum hydrocarbon, the microbial inoculum A or the microbial inoculum B is used for treating the material containing the petroleum hydrocarbon. The petroleum hydrocarbon-containing material includes petroleum hydrocarbon-contaminated soil or water. The petroleum hydrocarbon comprises normal paraffin, branched paraffin, cyclane and aromatic hydrocarbon. The invention takes the n-hexadecane as an experimental object to identify the degradation effect of the bacterial strain on the petroleum hydrocarbon.
The invention adopts yeast as the chassis cell for research and constructs the recombinant strain capable of degrading petroleum hydrocarbon. Based on the characteristics of clear genetic background of yeast and convenient molecular operation, the recombinant strain is simpler and easier to construct and obtain. In the invention, after genes for degrading alkane are introduced into saccharomyces cerevisiae and successfully expressed, the petroleum hydrocarbon is studied by using strains with higher degradation rate, and the degradation conditions are further optimized, so that the degradation efficiency can reach 91.63%.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Wherein, the three pseudomonas aeruginosa strains are all from the marine microorganism strain preservation management center and can be consulted with the relevant information of https:// mcc.
The strain number is as follows: 1a00364, central abbreviation: MCCC, chinese name: pseudomonas aeruginosa, the name of latin: pseudomonas, latin species name: aeruginosa, platform resource number: 1535C 0001000001066.
The strain number is as follows: 1a01151, center abbreviation: MCCC, chinese name: pseudomonas aeruginosa, the name of latin: pseudomonas, latin species name: aeruginosa, platform resource number: 1535C 0001000002415.
The strain number is as follows: 1a06466, central abbreviation: MCCC, chinese name: pseudomonas aeruginosa, the name of latin: pseudomonas, latin species name: aeruginosa, platform resource number: 1535C 0001000011941.
The invention is further illustrated by the following examples:
example 1
1. Solid plate verification that Saccharomyces cerevisiae BY474 cannot utilize n-hexadecane
Culture of Saccharomyces cerevisiae BY474 to OD600Is 0.5, is diluted by 10 times, 100 times and 1000 times in sequence, and the components of the culture medium are as follows in sequence: SC, SC-glucose +4g/L Tween 40+4g/L n-hexadecane. The culture was carried out in an incubator at 30 ℃ for 5 days. The results show that Saccharomyces cerevisiae BY4741 cannot use n-hexadecane as a carbon source.
2. Liquid fermentation verification that saccharomyces cerevisiae BY4741 cannot utilize n-hexadecane
Culture of Saccharomyces cerevisiae BY474 to OD6000.2, the culture medium used was: SC-glucose +4g/L Tween 40+4g/L hexadecane, the 96-hour growth curve of which is shown in 1-b in FIG. 1, can not grow basically. Then, the degradation rate of the n-hexadecane is measured, and the degradation rate is 0 after the fermentation for 4 days.
From the above data, it was concluded that: wild-type Saccharomyces cerevisiae BY4741 cannot use n-hexadecane as a carbon source.
Example 2 construction of n-hexadecane-degrading Strain
First, BY474 is taken as a basidiomycete
Three genes SYHY (the nucleic acid sequence is shown as SEQ ID NO:1 after codon optimization), AlmA (the nucleic acid sequence is shown as SEQ ID NO:2 after codon optimization) and alkm (the nucleic acid sequence is shown as SEQ ID NO:3 after codon optimization) with alkane degrading function are respectively selected and introduced into Saccharomyces cerevisiae BY 4741. The degradation scheme of the n-hexadecane is shown in figure 2. Genes SYHY, AlmA and alkm are integrated on a plasmid through a plasmid vector pRS416 to construct vectors pRS416-SYHY, pRS416-AlmA and pRS 416-alkm. The plasmids are respectively led into the underplate cell BY4741 to obtain three engineering modified strains which are respectively named as: SYHY, AlmA, almm. And carrying out fermentation verification on the obtained three strains:
four groups of strains are involved, namely wild type BY4741, strain SYHY, strain AlmA and strain almm. The medium used was SC-glucose +4g/L Tween 40+4g/L n-hexadecane, and the results are shown in FIG. 3: from the results, the degradation rate of the control group BY4741 in 96 hours of fermentation was 0, and the degradation rate of the strain AlmA in 96 hours was 14.48% among the three experimental strains SYHY, AlmA and almm, which were the highest degradation rate among the three.
Secondly, synthetic yeast containing only X chromosome is Chassis bacteria
Three genes SYHY, AlmA and almm with alkane degrading function are selected and respectively introduced into synthetic yeast only containing chromosome X (the strain is from Tianjin university).
Carrying out yeast genome rearrangement for screening:
8 single colonies transformed with SYHYX gene are selected and named as SYHYX-1, SYHYX-2, SYHYX-3, SYHYX-4, SYHYX-5, SYHYX-6, SYHYX-7 and SYHYX-8;
selecting 16 single colonies transformed with AlmA genes, and naming the single colonies as AlmAX-1, AlmAX-2, AlmAX-3, AlmAX-4, AlmAX-5, AlmAX-6, AlmAX-7, AlmAX-8, AlmAX-9, AlmAX-10, AlmAX-11, AlmAX-12, AlmAX-13, AlmAX-14, AlmAX-15 and AlmAX-16;
11 single colonies transformed with the akm gene were picked and named as akmX-1, akmX-2, alkmX-3, alkmX-4, alkmX-5, alkmX-6, alkmX-7, alkmX-8, alkmX-9, alkmX-10, and alkmX-11.
Further, the strains into which only the introduced gene was not rearranged in genome were designated as SYHYX-0, AlmAX-0 and alkmX-0.
The culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane. The strain was inoculated into the medium and the growth and degradation results were recorded as shown in figure 4:
as can be seen from the results of a and b in FIG. 4, the degradation rates of n-hexadecane in 96h fermentation of SYHYX-2 and SYHYX-4 in the strains obtained by screening through gene SYHY introduced into synthetic yeast for gene rearrangement are 11.37% and 10.35%, respectively. And from the growth condition, SYHYX-2 and SYHYX-4 have better growth vigor in the screened strains, and the OD is 72h600About 1.7. Compared with control group strains SYHYX-0, SYHYX-2 and SYHYX-4The degradation rate of the alkane is about 3 percent, which indicates that the strain with high degradation rate can be screened by using the gene rearrangement technology.
As can be seen from the results of c and d in FIG. 4, the strains obtained by introducing the gene AlmA into the synthetic yeast to carry out gene rearrangement and screening the strains with the highest degradation rates of the n-hexadecane, namely AlmAX-4 and AlmAX-8, during 96h fermentation, are 13.98% and 13.41%, respectively. And from the growth aspect, AlmAX-4 and AlmAX-8 are better in the screened strains for 72h, OD600About 1.7. Overall, it grew significantly better than most strains. Compared with the control group strain AlmAX-0, the degradation rate of the n-hexadecane of the AlmAX-4 and AlmAX-8 is about 7.5 percent higher, which is about 2 times that of the control group strain AlmAX-0.
As is clear from the results of e and f in FIG. 5, the strains obtained by introducing the gene alkkm into the synthetic yeast and carrying out gene rearrangement and screening the strains which have the highest degradation rates of n-hexadecane at 96h fermentation time, namely, alkmX-2 and alkmX-8, are 14.05% and 13.91%, respectively. And from the growth situation, the OD of alkmX-2 is 72h600OD of about 2, and alkmX-8600About 2.5, better growth in the selected strains, OD at other time points600Higher than most strains.
Compared with the degradation rate of 96h of n-hexadecane of the control group strain alkmX-0, the degradation rate of the n-hexadecane of the alkmX-2 and alkmX-8 is about 7 percent higher, which is about 2 times of the control group strain alkmX-0.
The degradation rate of the n-hexadecane of the starting strain X at 96h is 0, namely the starting strain X cannot utilize the n-hexadecane, and the growth curve OD of the starting strain X is600Less than the initial OD6000.2, which also confirms from the other side that the starting strain X cannot utilize n-hexadecane.
And (3) combining the results, selecting the strain with the highest degradation rate of 96h of n-hexadecane from the selected strains: the strain transformed with AlmA gene (strain AlmA) using BY4741 as Chassis strain was used as the strain for the next experiment.
Example 3 increasing the efficiency of the Strain's degradation of Petroleum hydrocarbons (exogenous addition of surfactant)
Respectively selecting a surfactant: rhamnolipid (R), sophorolipid (S), Tween 40(Tween 40, T) were used as subjects.
1. Effect of a Single surfactant on degradation efficiency
The experiments were divided into the following groups:
the strain is BY4741, and the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
② the strain is AlmA, the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
③ the strain is AlmA, SC-glucose, 300mg/L rhamnolipid, 4g/L Tween 40, 4g/L n-hexadecane
The strain is AlmA, SC-glucose, 300mg/L sophorolipid, 300mg/L rhamnolipid and 4g/L n-hexadecane
The strain is AlmA, SC-glucose, 300mg/L sophorolipid, 4g/L Tween 40, 4g/L n-hexadecane
As seen from a in FIG. 5, the growth curve of the wild type strain BY4741 was always flat, indicating that BY4741 could not grow; from b in FIG. 5, it is seen that the degradation rate of 96h n-hexadecane of BY4741 is 0, so both indicate that BY4741 cannot utilize n-hexadecane. When a single surfactant is exogenously added, the growth condition is shown in a figure 5 that 300mg/L sophorolipid is approximately equal to 300mg/L rhamnolipid which is more than 4g/L tween 40 is added and no surfactant is added. When 300mg/L sophorolipid and 300mg/L rhamnolipid are exogenously added, the growth conditions are almost consistent, and OD is 72h later600Can reach about 1.7, and is obviously better than adding 4g/L of Tween 40.
As shown by the b 96h degradation rate in FIG. 5, 300mg/L rhamnolipid > 300mg/L sophorolipid > 4g/L Tween 40 > is not added with a surfactant. And the highest degradation rate of the n-hexadecane is 18.45 percent when the rhamnolipid is added for 300mg/L for 96 h. The degradation rate is not much different from that of 96h after 300mg/L of sophorolipid is added, but the degradation rate of the n-hexadecane is improved by 8.06 percent and is nearly 2 times compared with that of the n-hexadecane without the surfactant. Therefore, when a single surfactant is added, the rhamnolipid of 300mg/L has the best effect.
2. Effect of different concentration gradient surfactants on hexadecane degradation
All strains are AlmA, and the experiment is divided into the following groups:
1-1: the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
1-2: the culture medium comprises SC-glucose, 100mg/L sophorolipid and 4g/L n-hexadecane
1-3: the culture medium comprises SC-glucose, 200mg/L sophorolipid and 4g/L n-hexadecane
1-4: the culture medium comprises SC-glucose, 300mg/L sophorolipid and 4g/L n-hexadecane
1-5: the culture medium comprises SC-glucose, 400mg/L sophorolipid and 4g/L n-hexadecane
2-1: the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
2-2: the culture medium is SC-glucose, 100mg/L rhamnolipid and 4g/L n-hexadecane
2-3: the culture medium is SC-glucose, 200mg/L rhamnolipid and 4g/L n-hexadecane
2-4: the culture medium is SC-glucose, 300mg/L rhamnolipid and 4g/L n-hexadecane
2-5: the culture medium is SC-glucose, 400mg/L rhamnolipid and 4g/L n-hexadecane
As shown by a in FIG. 6, the growth curve shows that the exogenous addition of 300mg/L sophorolipid > 200mg/L sophorolipid > 400mg/L sophorolipid > 100mg/L sophorolipid > does not add any surfactant. As shown by b in FIG. 6, the degradation rate of 96h n-hexadecane is shown, and the degradation rate of 200mg/L sophorolipid added from outside and that of 300mg/L sophorolipid are both greater than 17%, and the difference between the degradation rates is not great. The degradation rate is sequentially higher than that of the externally added sophorolipid of 400mg/L, higher than that of the externally added sophorolipid of 100mg/L and higher than that of the externally added sophorolipid without any surfactant. And the degradation rate of the n-hexadecane is the highest when 300mg/L of sophorolipid is exogenously added, the highest degradation efficiency is 17.86 percent, the degradation rate of the n-hexadecane is improved by 7.46 percent compared with the degradation rate of the n-hexadecane when the sophorolipid is not added, and the degradation rate of the n-hexadecane is improved by 4.03 percent compared with the degradation rate of the n-hexadecane when 100mg/L of sophorolipid is exogenously added, but the degradation rate is almost the same as the degradation rate of the n-hexadecane when 200mg/L of sophorolipid is exogenously added, and the difference is only 0.72 percent. Therefore, it can be said that the exogenous addition of 200mg/L sophorolipid is the same as, and the most effective is the exogenous addition of 300mg/L sophorolipid. As shown by c in FIG. 6, the growth curve shows that 300mg/L rhamnolipid > 400mg/L rhamnolipid > 200mg/L rhamnolipid > 100mg/L rhamnolipid > is exogenously added without any surfactant. As shown in d in FIG. 6, the degradation rate of 96h of n-hexadecane is shown, the degradation rate of n-hexadecane is the highest when 300mg/L of rhamnolipid is added externally, the highest degradation efficiency is 18.45%, and the degradation rate of n-hexadecane is about 16% when 200mg/L of rhamnolipid and 400mg/L of rhamnolipid are added externally after 96h of n-hexadecane. Compared with the rhamnolipid added by 100mg/L from an external source, the effect is obvious, and the degradation rate of n-hexadecane is improved by 8.05 percent when compared with the condition that the rhamnolipid is not added.
3. Effect of surfactant combinations on hexadecane degradation
By exploring exogenously added pairwise combined surfactants or tri-combined surfactants, the degradation rate of n-hexadecane is improved to different degrees:
3.1 the experiments were divided into the following groups:
the method comprises the following steps: the strain is AlmA, the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
Secondly, the step of: the strain is AlmA, the culture medium is SC-glucose +300mg/L rhamnolipid +4g/L Tween 40+4g/L n-hexadecane
③: the strain is AlmA, and the culture medium is SC-glucose, 300mg/L sophorolipid, 300mg/L rhamnolipid and 4g/L n-hexadecane
Fourthly, the method comprises the following steps: the strain is AlmA, the culture medium is SC-glucose, 300mg/L sophorolipid, 4g/L Tween 40 and 4g/L n-hexadecane
Fifthly: the strain is BY4741, and the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
As seen from the growth curve a in FIG. 7, OD of the wild type strain BY4741600Hardly changes; as seen from the degradation rate of 96h n-hexadecane shown in b in FIG. 7, the degradation rate of 96h n-hexadecane of BY4741 was 0. When the surfactant is added in pairs by external sources, from the growth condition of b in figure 7, the growth of 300mg/L sophorolipid +4g/L tween 40 is better than that of 300mg/L rhamnolipid +4g/L tween 40 is better than that of 300mg/L sophorolipid +300mg/L rhamnolipid is better than that of a control group without the surfactant. Shown as b in FIG. 7, 96h plus tenThe degradation rate of the hexaalkane is the highest in the case of 300mg/L sophorolipid +4g/L tween 40, then 300mg/L rhamnolipid +4g/L tween 40, and finally 300mg/L sophorolipid +300mg/L rhamnolipid, which is consistent with the growth curve. And the degradation rate of 300mg/L sophorolipid +4g/L Tween 40 is 23.34%, which is about 13% higher than that of the control group without adding surfactant, and is more than 2 times thereof. Therefore, when the surfactant is combined two by two, the optimal combination is 300mg/L sophorolipid +4g/L Tween 40.
3.2 the experiments were divided into the following groups:
the method comprises the following steps: the strain is AlmA, the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
Secondly, the step of: the strain is AlmA, the culture medium is SC-glucose, 300mg/L sophorolipid, 300mg/L rhamnolipid, 4g/L Tween 40, 4g/L n-hexadecane
③: the strain is BY4741, and the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
As seen from the a-growth curve in FIG. 8, OD of the wild type strain BY4741600Hardly changes; OD of the three-component surfactant 300mg/L sophorolipid, 300mg/L rhamnolipid and 4g/L tween 40 when added externally600The OD of the product is obviously higher than that of a control group without any surfactant, and the OD of the product is 96 hours6002.1, OD compared with OD without addition of surfactant600The increase is about 1.8. As can be seen from b in fig. 9, the degradation rate of n-hexadecane for 96h is shown, when the tri-combination surfactant is added externally, the degradation rate of n-hexadecane for 96h is 23.52%, which is higher than that of the control group without any surfactant. Therefore, when the three-combination surfactant 300mg/L sophorolipid, 300mg/L rhamnolipid and 4g/L tween 40 are added externally, the 96-hour degradation rate of the n-hexadecane can be obviously improved.
3.3 to sum up, the degradation rate of hexadecane when a single surfactant, two combined surfactants and three combined surfactants are added is compared, and the result is as follows:
the experiments were divided into the following groups:
the method comprises the following steps: the strain is BY4741, and the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
Secondly, the step of: the strain is AlmA, the culture medium is SC-glucose +4g/L Tween 40+4g/L n-hexadecane
③: the strain is AlmA, the culture medium is SC-glucose, 300mg/L sophorolipid and 4g/L n-hexadecane
Fourthly, the method comprises the following steps: the strain is AlmA, the culture medium is SC-glucose, 300mg/L sophorolipid, 4g/L Tween 40 and 4g/L n-hexadecane
Fifthly: the strain is AlmA, the culture medium is SC-glucose, 300mg/L sophorolipid, 300mg/L rhamnolipid, 4g/L Tween 40, 4g/L n-hexadecane
As seen from the growth curve a in FIG. 9, OD of the wild type strain BY4741600Hardly changes; the growth of the exogenously added three-combined surfactant of 300mg/L sophorolipid, 300mg/L rhamnolipid and 4g/L tween 40 and the exogenously added 300mg/L sophorolipid plus 4g/L tween 40 is almost consistent before 72h, 72h to 96h, the OD of the three-combined surfactant600The ratio is obviously higher than that of two combined surfactants, namely 300mg/L sophorolipid plus 4g/L Tween 40, OD600Can reach more than 2.2. Moreover, the growth of the two is obviously better than that of the sophorolipid added with a single surfactant of 300 mg/L. As shown in the b in FIG. 9, the 96h degradation rate of n-hexadecane is 23.52%, and the highest 96h degradation rate of n-hexadecane is obtained when the three combination surfactants are added from the outside. However, the degradation rate of the two combined surfactants of externally added 300mg/L sophorolipid and 4g/L Tween 40 can also reach 23.34%, and the degradation rates are almost the same. And both the two are obviously higher than the two obtained by adding 300mg/L sophorolipid of a single surfactant, and compared with a control group, the degradation rate is improved by 1 time.
Example 4 increasing the efficiency of the degradation of Petroleum hydrocarbons by the strains (mixed with Pseudomonas aeruginosa)
When the surfactant is exogenously added, the degradation rate of hexadecane can be obviously improved, so from the viewpoint of constructing a mixed bacteria system, three pseudomonas aeruginosa strains, namely 1A00364, 1A01151 and 1A06466 are introduced, and the three pseudomonas aeruginosa strains can generate the surfactant by utilizing the hexadecane.
1. Hexadecane is degraded by three pseudomonas aeruginosa strains alone
The experiments were divided into the following groups:
firstly, the strain is pseudomonas aeruginosa 1A00364, and the culture medium is liquid LB culture medium +4g/L n-hexadecane;
secondly, the strain is pseudomonas aeruginosa 1A01151, and the culture medium is a liquid LB culture medium and 4g/L n-hexadecane;
③, the strain is pseudomonas aeruginosa 1A06466, and the culture medium is liquid LB culture medium +4g/L n-hexadecane.
As shown in a in FIG. 10, the growth curves of three P.aeruginosa plants showed that the three P.aeruginosa plants were almost uniformly grown before 12 hours, and OD was from 12 hours to 48 hours600The values are 1A01151 > 1A00364 > 1A06466 in sequence from large to small. And 1A01151 at 36h to 48h, OD600About 2.5. As shown in b in figure 11, the degradation rate of 48h n-hexadecane of three pseudomonas aeruginosa is the highest in the degradation rate of 71.34% in the genus 1A06466, which is higher than that of 1A00364 and 1A 01151. Therefore, three pseudomonas aeruginosa strains can efficiently degrade the n-hexadecane, and the degradation rates of the three pseudomonas aeruginosa strains are not very different.
2. Three pseudomonas aeruginosa (1A00364, 1A01151 and 1A06466) have small difference in degradation rate for 48 hours, so that the three pseudomonas aeruginosa and the strain AlmA obtained by engineering transformation are selected to construct a mixed strain system. The results of preliminary exploration for the case of using n-hexadecane in the novel mixed culture system are shown in FIG. 11:
as can be seen from FIG. 11, in the three combinations of AlmA +1A00364, AlmA +1A01151 and AlmA +1A06466, the combination of AlmA +1A06466 has the largest degradation rate of 48h n-hexadecane, which can reach 79.80%. At this time, the degradation efficiency of the two-bacterium system is greater than the arithmetic sum of the degradation efficiencies of the two bacteria alone, and the effect of "1 +1 > 2" is realized. And the degradation rate of the three combinations of 48h of n-hexadecane is very small, and is only about 5%. However, it is significantly higher than the single engineered strain AlmA, which has a degradation rate of only 4.57% of n-hexadecane at 48 h.
3. The combination of AlmA +1A06466 is used as a research object of a mixed bacteria system to optimize fermentation conditions so as to improve the degradation rate of n-hexadecane. The results are shown in figure 12 of the drawings,
3.1 setting inoculation ratio of 5 gradients, 1A 06466: the AlmA is respectively 4:1, 2:1, 1:2 and 1:4,and searching the optimal inoculation ratio. Initial OD in AlmA600Set to 0.2, initial OD of P.aeruginosa 1A064666000.8, 0.4, 0.2, 0.1 and 0.05 in sequence. As can be seen from a in FIG. 12, when the inoculation ratio was 2:1, the degradation rate of n-hexadecane was 84% at the highest in 48 hours. When the inoculation ratio is more than or less than 2:1, the degradation rate of the n-hexadecane is less than 2:1 after 48 hours. Therefore, the optimal ratio of inoculation is 2: 1.
3.2 the optimal growth temperature of the saccharomyces cerevisiae is 30 ℃, and the growth temperature range of the pseudomonas aeruginosa is 25-42 ℃, so that the research selects five temperature gradients of 24 ℃, 27 ℃, 30 ℃, 33 ℃ and 36 ℃ for experiments. From the results b in FIG. 12, it is understood that the efficiency of degrading n-hexadecane by the mixed bacteria system is the highest at 79.8% when the temperature is 30 ℃, and that the influence of different temperatures on degrading n-hexadecane by the mixed bacteria system is very different. The degradation rate of 48h of n-hexadecane is more than 50% at 24 ℃ and 27 ℃, and the degradation rate is different from 30% at 30 ℃. The degradation rate of 48h of n-hexadecane is about 40% at 33 ℃ and 36 ℃, and the difference between the degradation rate and the highest degradation rate is 40%. Therefore, the optimum fermentation temperature is 30 ℃.
3.3 besides the temperature can significantly affect the degradation rate of petroleum hydrocarbons, the pH value of the fermentation broth, i.e. pH, can also affect the degradation rate of n-hexadecane. In this study, 5 pH gradients of 4.5, 5.5, 6.5, 7.5 and 8.5 were selected to explore the optimal pH. As can be seen from c in FIG. 12, the mixed culture system degraded n-hexadecane with the highest efficiency of 82.95% in 48h at pH 7.5. When the pH value is lower than 7.5, the degradation rate of the n-hexadecane is increased along with the increase of the pH value for 48 h; the degradation rate of 48h n-hexadecane was instead lower at pH 8.5 than at pH 8.5. Therefore, the optimum pH is 7.5.
3.4 the better the P.aeruginosa grows using n-hexadecane to produce rhamnolipid, the faster the n-hexadecane is consumed and the more rhamnolipid is produced. The more rhamnolipid is generated, the more viscosity can be reduced, the fluidity can be increased, the contact with cells can be increased, and the degradation rate of n-hexadecane can be further improved. NaCl plays an important role in maintaining the osmotic pressure of cell growth, so the research explores the influence of NaCl with different concentrations on the degradation of n-hexadecane. In the study, 5 NaCl concentration gradients were set, 6, 8, 10, 12, and 14g/L, respectively. As seen from d in FIG. 12, the degradation rate of n-hexadecane was 83.6% at 48 hours when the NaCl concentration was 10 g/L. And the degradation rates of the 5 concentration gradients are all concentrated between 73 percent and 83 percent, and the degradation rates are not greatly different.
3.5 through optimization of fermentation conditions, the inoculation ratio is 1A 06466: the results of fig. 13 show that the degradation rate of n-hexadecane in 48 hours after the fermentation condition was optimized was increased by 12% compared with that before the fermentation condition was optimized, when the two-strain system degraded n-hexadecane in 48 hours, the results were obtained when AlmA is 2:1, 30 ℃, the pH of the medium is 7.5, the NaCl concentration is 10 g/L.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
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<120> recombinant strain, composite strain and petroleum hydrocarbon biodegradation method
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agagctaaga ttgccaaggg tactgataac atcaacggta ag 882

Claims (17)

1. A recombinant yeast expressing an alkane-degrading gene;
the alkane degrading genes include: SYHY gene, AlmA gene or almm gene.
2. The recombinant yeast according to claim 1,
the bottom plate bacterium is Saccharomyces cerevisiae or synthetic yeast containing only chromosome X;
the nucleic acid sequence of SYHY gene is shown as SEQ ID NO. 1;
the nucleic acid sequence of the AlmA gene is shown as SEQ ID NO. 2;
the nucleic acid sequence of the alk gene is shown as SEQ ID NO. 3.
3. The recombinant yeast according to claim 2, wherein the chassis bacteria are saccharomyces cerevisiae BY 4741; the gene expresses AlmA gene of a nucleic acid sequence shown as SEQ ID NO. 2.
4. A method for constructing a recombinant yeast according to any one of claims 1 to 3, comprising: the alkane degrading gene is introduced into yeast.
5. The method of claim 4, wherein the introducing comprises constructing a plasmid vector containing the alkane degrading gene, transforming into yeast; the plasmid vector is an integrative plasmid vector.
6. The method of claim 5, wherein the integrative plasmid is pRS416, and the insertion site of the alkane degradation gene on the plasmid vector is between Hind III and BamH I.
7. Use of the recombinant yeast of any one of claims 1 to 3 for degrading petroleum hydrocarbons.
8. A petroleum hydrocarbon degrading agent A comprising the recombinant yeast according to any one of claims 1 to 3.
9. The microbial agent A according to claim 8, further comprising a culture medium and/or a surfactant; the culture medium is an SC culture medium; the surfactant is at least one of rhamnolipid, sophorolipid and tween.
10. A petroleum hydrocarbon degrading microbial inoculum B comprising the recombinant yeast of any one of claims 1 to 3 and pseudomonas aeruginosa.
11. The microbial inoculum B of claim 10, wherein the quantity ratio of the recombinant yeast to the pseudomonas aeruginosa is (1-4): (1-4).
12. The microbial inoculum B of claim 10 or 11, wherein the Pseudomonas aeruginosa is 1A00364, 1A01151 or 1A 06466.
13. The microbial agent B according to any one of claims 8 to 12, further comprising an LB medium in which the NaCl concentration is 10g/L and the pH value is 7.5.
14. A method for degrading petroleum hydrocarbon, characterized in that the petroleum hydrocarbon is degraded by the microbial inoculum A of claim 8 or 9 or the microbial inoculum B of any one of claims 10 to 13.
15. The method according to claim 14, characterized in that the degradation is carried out with the agent a:
in the degradation system, the initial OD of the microbial inoculum A600The value is 0.2, the concentration of the petroleum hydrocarbon is 4g/L, and the concentration of the surface active agent is 300 mg/L-4 g/L;
the degradation conditions comprise that the temperature is 30 ℃, and the vibration degradation is carried out at 220rpm for 3-4 days.
16. The method according to claim 14, characterized in that the degradation is carried out with the agent B:
in the degradation system, the initial OD of the microbial inoculum B600The value is 0.2, and the concentration of the petroleum hydrocarbon is 4 g/L;
the degradation conditions comprise that the temperature is 30 ℃, and the vibration degradation is carried out at 220rpm for 3-4 days.
17. The method of any one of claims 14 to 16, wherein the petroleum hydrocarbon comprises n-hexadecane.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114807182A (en) * 2022-06-07 2022-07-29 天津大学 Yeast strain for degrading long-chain alkane and application thereof
CN114891649A (en) * 2022-06-07 2022-08-12 天津大学 Composite bacteria and application thereof in degradation of long-chain alkane

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110846257A (en) * 2019-12-11 2020-02-28 中冶华天工程技术有限公司 Microbial bacterium for degrading long-chain alkane and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110846257A (en) * 2019-12-11 2020-02-28 中冶华天工程技术有限公司 Microbial bacterium for degrading long-chain alkane and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HANANO,A ET AL.: "Accession NO.AGZ83172.1,alkane-inducible cytochrome P450 52A3(Saccharomyces cerevisiae", 《GENBANK DATABASE》 *
张爱君等: "海洋石油降解菌的筛选、鉴定及降解活性", 《化学工业与工程》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114807182A (en) * 2022-06-07 2022-07-29 天津大学 Yeast strain for degrading long-chain alkane and application thereof
CN114891649A (en) * 2022-06-07 2022-08-12 天津大学 Composite bacteria and application thereof in degradation of long-chain alkane
CN114807182B (en) * 2022-06-07 2023-07-25 天津大学 Yeast strain for degrading long-chain alkane and application thereof

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