CN113773973A - Yeast capable of tolerating high-concentration furfural and application thereof - Google Patents
Yeast capable of tolerating high-concentration furfural and application thereof Download PDFInfo
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- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 title claims abstract description 136
- 240000004808 Saccharomyces cerevisiae Species 0.000 title abstract description 13
- 239000000413 hydrolysate Substances 0.000 claims abstract description 26
- 241000222178 Candida tropicalis Species 0.000 claims abstract description 23
- 238000004321 preservation Methods 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims description 2
- 238000009395 breeding Methods 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 14
- 230000004151 fermentation Effects 0.000 abstract description 14
- 230000012010 growth Effects 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 5
- 108010009736 Protein Hydrolysates Proteins 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 2
- 230000000593 degrading effect Effects 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 30
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 20
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 238000009630 liquid culture Methods 0.000 description 11
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 10
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 10
- 238000012258 culturing Methods 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 239000008103 glucose Substances 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 6
- XPFVYQJUAUNWIW-UHFFFAOYSA-N furfuryl alcohol Chemical compound OCC1=CC=CO1 XPFVYQJUAUNWIW-UHFFFAOYSA-N 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 6
- 239000000811 xylitol Substances 0.000 description 6
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 6
- 229960002675 xylitol Drugs 0.000 description 6
- 235000010447 xylitol Nutrition 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 230000003044 adaptive effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000221523 Rhodotorula toruloides Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 3
- 238000007598 dipping method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000012978 lignocellulosic material Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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Abstract
The invention discloses a yeast strain capable of tolerating high-concentration furfural. The strain is candida tropicalis (Candida tropicalis) Y31-N, which has been preserved in China Center for Type Culture Collection (CCTCC) at 2/3/2021 with the preservation number of CCTCC M2021180. The strain is original candida tropicalis (preserved by experiments)Candida tropicalisAS 2.1776) is continuously domesticated, and agricultural and forestry residue hydrolysate (short for: hydrolysate) with strong toleranceCandida tropicalisY31-N. Screened by the inventionCandida tropicalisCompared with original candida tropicalis, the strain has large furfural tolerance and utilization capacityThe method has the advantages of greatly improving the growth rate, being capable of growing in the hydrolysate containing 6.05 g/L of furfural without detoxifying the hydrolysate, completely degrading the furfural in the hydrolysate within 6-8 h, and not obviously inhibiting the growth and fermentation capacity. The excellent characteristics of the strain enable the strain to have good industrial application prospect, and reference is provided for solving the problem that furfural in hydrolysate inhibits the growth of microorganisms.
Description
Technical Field
The invention relates to the field of biochemical engineering, in particular to saccharomycetes capable of tolerating high-concentration furfural and application thereof.
Background
The major component of the agricultural and forestry residues is lignocellulosic material, widely available, mainly comprising cellulose, hemicellulose and lignin, which is a renewable, attractive and promising source of fermentation substrates that can be used to produce biofuels (e.g., cellulosic ethanol) and high value-added chemicals (e.g., xylitol). Lignocellulosic material contains a large amount of hemicellulose and cellulose, a carbohydrate polymer composed primarily of xylose and glucose units, which can be hydrolyzed to produce xylose and glucose solutions, which can then be used as a fermentation medium to obtain ethanol, xylitol, and other useful products. In order to release carbon sources such as glucose, xylose and the like which can be used for microbial growth and fermentation in the lignocellulose material, agricultural and forestry residue raw materials need to be subjected to hydrolysis treatment, but by-products such as furfural, acetic acid and phenol which can strongly inhibit the microbial growth and fermentation are generated in the hydrolysis treatment process. Furfural is one of typical inhibitors in hydrolysate, is dehydrated from xylose, and is important to develop strains capable of resisting the inhibitors in the hydrolysate in order to avoid time consumption and pollution to the detoxification process of agriculture and forestry remainder hydrolysate (hydrolysate for short).
A number of microorganisms have been developed which are tolerant to lignocellulosic hydrolysates, such as E.coli (E.coli) (II)Escherichia coli) Saccharomyces cerevisiae (A)Saccharomyces cerevisiae) Rhodosporidium toruloides (A) and Rhodosporidium toruloides (B)Rhodosporidium toruloides). The research on the furfural tolerance mechanism of yeast mainly focuses onSaccharomyces cerevisiaeAnd the research on the tolerance mechanism of industrial yeast with high tolerance is less, so that the deep understanding and the practical application of the furfural pressure response mechanism of yeast cells are hindered.
Strategies for increasing the tolerance of microorganisms to hydrolysate by-products mainly include: genetic manipulation and adaptive evolution of genes. Compared with the genetic manipulation technology for reasonably designing the strains, the adaptive evolution technology has the advantages that the cost of the evolution engineering is low, the obtained strains have stable inheritance, and the heterogeneous protein expression which generates metabolic burden and needs strict expression control is not introduced.
Based on the advantages of the adaptive evolution engineering technology, the invention adopts an adaptive domestication method to separate the hydrolysate containing the inhibitory compound furfural and having stronger toleranceCandida tropicalisY31-N. Compared with the original Candida tropicalis strain, the tolerance and furfural utilization capability of the strain is greatly improved, and 6.05 g/L furfural can be completely degraded within 6-8 h. The excellent characteristics of the strain enable the strain to have good industrial application prospect, and support can be provided for solving the problems that furfural in hydrolysate inhibits the growth of microorganisms and reduces the fermentation performance.
Disclosure of Invention
The invention provides a yeast capable of tolerating high-concentration furfural, namely Candida tropicalis (preserved by experiment)Candida tropicalisCGMCC 2.1776) is continuously domesticated to separate out furfural with strong tolerance to inhibitory compound in lignocellulose hydrolysateCandida tropicalis Y31-N. The obtained domesticated strain can tolerate high-concentration furfural and can normally grow in hydrolysate with furfural concentration as high as 6.05 g/L.
The yeast strain(s) capable of tolerating high-concentration furfuralCandida tropicalis) Y31-N, which has been preserved in China Center for Type Culture Collection (CCTCC) at 2/3/2021 with the preservation number of CCTCC M2021180.
The high-concentration furfural-tolerant yeast strain is characterized in that the domestication is carried out in a mode of increasing the concentration of furfural in a gradient manner.
The high-concentration furfural-tolerant yeast strain specifically comprises the following steps:
1) and (3) activation: selecting a single colony on a solid preservation culture medium plate, inoculating the single colony into a liquid culture medium without furfural, and culturing for 12-24 h;
2) domestication and screening: inoculating the activated bacterial liquid into a hydrolysis liquid culture medium containing furfural at 6-12% of inoculation amount, and culturing at 28-35 deg.C to obtain initial OD600=0.9-1.1, when the culture reaches the exponential phase, transferring the culture medium to a corresponding solid culture medium with the same furfural concentration, and culturing the culture medium to 36-54 h. And then selecting good colonies which grow fast and are large in individual size to a liquid culture medium without furfural, culturing to an exponential phase, and transferring to a hydrolysate culture medium with higher furfural concentration for second round of domestication culture. Repeatedly carrying out multiple passages, wherein the passage times are 20 or more than 20, selecting excellent strains which have good growth vigor and can tolerate 6.05 g/L furfural and are large in size, carrying out slant preservation in a solid preservation culture medium, and sealing and culturing in a constant-temperature incubator at 28-33 ℃ for 36-54 h; washing the inclined plane with 18-22% sterile glycerol, packaging the washing solution in a bacteria-protecting tube, and freezing and preserving at-80 deg.C to obtain domesticated yeastCandida tropicalis Y31-N。
The solid preservation culture medium comprises the following components: 15-25 g/L of xylose, 2-14 g/L of glucose, 3-7 g/L of peptone, 3-8 g/L of yeast extract powder and KH2PO4 3-6 g/L，MgSO40.2-0.5 g/L, 0.3-0.6 g/L ammonium sulfate, 18-25 g/L agar and natural pH.
The liquid culture medium without furfural comprises the following components: 15-25 g/L of xylose, 2-14 g/L of glucose, 3-7 g/L of peptone, 3-8 g/L of yeast extract powder and KH2PO4 3-6 g/L，MgSO40.2-0.5 g/L, 0.3-0.6 g/L ammonium sulfate, and natural pH.
The hydrolysis liquid culture medium containing the furfural mainly comprises the following components: 1.2-6.1 g/L of furfural, 42.2-102.8 g/L of xylose and 3.8-11.1 g/L, KH of glucose2PO4 3.1-5.2 g/L、MgSO4 
H2O 0.21-0.57 g/L、(NH4)2HPO4 2.1-3.5 g/L，pH 6-7。
The hydrolysis liquid solid culture medium without furfural mainly comprises the following components: 1.2-6.1 g/L of furfural, 42.2-102.8 g/L of xylose and 3.8-11.1 g/L, KH of glucose2PO4 3.1-5.2 g/L、MgSO4 
H2O 0.21-0.57 g/L、(NH4)2HPO42.1-3.5 g/L, agar 18-25 g/L, and pH 6-7.
The furfural-containing hydrolysate solid culture medium mainly comprises the following components: 1.2-6.1 g/L of furfural, 42.2-102.8 g/L of xylose and 3.8-11.1 g/L, KH of glucose2PO4 3.1-5.2 g/L、MgSO4 
H2O 0.21-0.57 g/L、(NH4)2HPO42.1-3.5 g/L, agar 18-25 g/L, and pH 6-7.
The yeast strain, wherein the liquid culture medium culture conditions of step 1) and step 2) are as follows: at 28-33 ℃, the rotation speed of the shaking table is 120 and 240 rpm.
The original strain capable of tolerating the high-concentration furfural yeast strain is preserved in a laboratoryCandida tropicalisCGMCC 2.1776), purchased at the institute of microbiology, department of chinese.
In another aspect of the invention, the application of the candida tropicalis in the aspect of tolerating high-concentration furfural is provided.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the Candida tropicalis capable of tolerating high-concentration furfural provided by the inventionCandida tropicalis Y31-N can normally grow in hydrolysate containing high-concentration furfural (6.05 g/L), the strain has strong environmental adaptability and better tolerance to furfural inhibitors, and furfural can be completely degraded within 6-8 h. The strain can be used as a furfural-tolerant strain in hydrolysate fermentation. The domesticated strain provides a good solution for growth and fermentation of non-detoxified hydrolysate, reduction of equipment investment, reduction of water consumption, reduction of cost, improvement of efficiency and increase of yield, and has important significance for promotion of industrialization of biomass conversion.
Detailed Description
Example 1 acclimatization of Candida tropicalis
The domestication method comprises the following steps:
(1) candida tropicalis (C.tropicalis) (C.tropicalis)Candida tropicalisCGMCC 2.1776) as a primary strain, wherein the Candida tropicalis primary strain is purchased from institute of microbiology of Chinese academy of sciences;
selecting an original strain single colony, inoculating the original strain single colony into a liquid culture medium without furfural, and culturing for 13-18 h; inoculating the activated bacterial liquid into a hydrolysis liquid culture medium containing furfural in an inoculation amount of 8-10% to ensure that the initial OD is6000.9-1.1, the culture temperature is 28-32 ℃, and the culture is carried out for 12-18 h, namely the primary culture bacterial liquid is obtained;
dipping the primary culture bacterial liquid by using an inoculating loop, dividing three areas on a flat plate of a hydrolysate solid culture medium containing furfural for inoculation, inversely culturing in a constant-temperature incubator at 30-32 ℃, picking out a single bacterial colony with better growth vigor on the flat plate after 24-54h, inoculating into a sterilized liquid culture medium without furfural, and performing shaking culture on a table for 12-16 h to obtain a secondary seed liquid; inoculating the secondary seed liquid into a fresh furfural-containing hydrolysate culture medium, wherein the inoculation amount is 8-12%, and culturing for 12-24 h to obtain a secondary culture bacterial liquid;
dipping the secondary culture bacterial liquid by using an inoculating loop, dividing three areas on a solid hydrolysate culture medium plate containing furfural and having higher furfural content for inoculation, carrying out inverted culture in a constant-temperature incubator at 30-32 ℃, picking a single bacterial colony with better growth vigor on the plate after 24-54h, inoculating the single bacterial colony into a sterilized liquid culture medium containing no furfural, and carrying out shaking culture on a shaker for 12-18 h to obtain a tertiary seed liquid;
inoculating the third seed solution into fresh furfural-containing hydrolysate with further increased content, inoculating the third seed solution at an amount of 8-12%, culturing for 12-20 h, performing shake culture on a shaker to obtain a third culture solution, repeating the steps until a twentieth generation culture solution is obtained;
under the aseptic condition, dipping the twentieth generation culture bacteria liquid by using an inoculating loop, carrying out three-area inoculation on a furfural-containing hydrolysate solid culture medium plate with the furfural concentration of 6.05 g/L, carrying out inverted culture in a constant-temperature incubator at 30-32 ℃, picking a single colony with better growth vigor on the plate after 24-54h, carrying out slant preservation in a solid preservation culture medium, sealing, and culturing in the constant-temperature incubator at 28-33 ℃ for 36-54 h; washing the inclined plane with 18-20% sterile glycerol, and mixing the washing solutionsSubpackaging in a bacteria-protecting tube, and freezing and preserving at-80 deg.C to obtain domesticated yeastCandida tropicalis Y31-N。
Example 2 domesticated YeastCandida tropicalisEvaluation of Furfural-resistant growth and fermentation Capacity of Y31-N
Compared with the original strain, the tolerant strain shows a shorter lag phase in a furfural-containing hydrolysate culture medium, and completely degrades furfural within 6-8 h, wherein the concentration of furfuryl alcohol is 3.1 g/L; domesticated yeastCandida tropicalisY31-N enters into exponential growth phase at 6-10 h and enters into stationary phase at 39-54 h, while the cell growth of the original strain almost completely stagnates in the initial growth stage in the furfural-containing hydrolysate culture medium, and does not gradually enter into exponential growth phase until 33-40 h, and the final biomass is 25-38% of the tolerant strain.
Moreover, compared with the original strain, the fermentation capacity evaluation is carried out in a hydrolysate culture medium containing furfural (the concentration of furfural is 6.05 g/L), the final xylitol yield of the tolerant strain is not obviously influenced, the conversion rate of xylitol is 41-59% after fermentation is carried out for 38-54 h, and the result difference with the culture medium containing no furfural under the same conditions is not obvious. And the original strain is fermented in a hydrolysis liquid culture medium containing furfural (the concentration of furfural is 6.05 g/L), under the same condition, compared with the result in a culture medium without furfural, the conversion rate of xylitol is reduced by 3-8 times, and the fermentation capacity is greatly reduced.
And (3) measuring the OD value of the diluted bacteria-containing fermentation culture solution by using an enzyme-labeling instrument at 600 nm, and detecting the relative content of the biomass.
The detection of the contents of xylose, xylitol, furfural and furfuryl alcohol in the fermentation liquor is carried out by adopting a high performance liquid chromatography, drawing a standard curve by using standard series contents to peak area, and carrying out content determination by using retention time for qualitative determination and peak area for quantitative determination.
Claims (4)
1. The candida tropicalis capable of tolerating high-concentration furfural is characterized in that the strain isCandida tropicalisY31-N, which has been preserved in China center for type culture Collection (CGMCC) at 2/3/2021The strain is CCTCC M2021180, and the original Candida tropicalis strain is purchased from the institute of microbiology in the department of China, and the preservation number is CGMCC 2.1776.
2. The high concentration furfural resistant Candida tropicalis Y31-N of claim 1 in which Candida tropicalis (C.tropicalis) (C.tropicalis)) (C.tropicalis)) is usedCandida tropicalisCGMCC 2.1776) as an initial strain, and screening the Candida tropicalis with high-concentration furfural resistance by an domestication breeding method.
3. Use of candida tropicalis according to claim 1 for highly efficient tolerance of high concentrations of furfural.
4. The Candida tropicalis with high furfural tolerance according to claim 3, wherein the strain can normally grow in the agricultural and forestry residue hydrolysate with high furfural content (6.05 g/L).
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| SU1463755A1 (en) * | 1986-12-24 | 1989-03-07 | Научно-Производственное Гидролизное Объединение "Гидролизпром" | Method of producing hydrodrolysis media for growing fodder yeast |
| CN101165166A (en) * | 2007-10-10 | 2008-04-23 | 首都师范大学 | Novel use for Candida tropicalis |
-
2021
- 2021-05-26 CN CN202110580108.2A patent/CN113773973B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1463755A1 (en) * | 1986-12-24 | 1989-03-07 | Научно-Производственное Гидролизное Объединение "Гидролизпром" | Method of producing hydrodrolysis media for growing fodder yeast |
| CN101165166A (en) * | 2007-10-10 | 2008-04-23 | 首都师范大学 | Novel use for Candida tropicalis |
Non-Patent Citations (2)
| Title |
|---|
| SHIZENG WANG ET AL: "Furfural tolerance and detoxification mechanism in Candida tropicalis", BIOTECHNOLOGY FOR BIOFUELS, vol. 9, no. 250, pages 1 - 11 * |
| SHIZENG WANG ET AL: "Xylose enhances furfural tolerance in Candida tropicalis by improving NADH recycle", CHEMICAL ENGINEERING SCIENCE, vol. 158 * |
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| CN113773973B (en) | 2024-02-23 |
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