Candida nivariegata GXAS-CN strain and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a Candida nivariensis strain GXAS-CN, with a deposit number of GDMCC No. 62755. The GXAS-CN strain has excellent antioxidant stress resistance, can tolerate the stress of high concentration hydrogen peroxide, has the characteristics of high temperature resistance and other fermentation inhibitors, and has good ethanol production capacity. Therefore, the present invention also provides the use of the GXAS-CN strain in bioethanol production.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to a Candida nivariegata GXAS-CN strain and application thereof. [Background technology]

[0002] Affected by energy shortages, environmental pollution and other issues, countries around the world are vigorously developing the bioethanol industry to reduce dependence on oil and reduce automobile exhaust emissions during economic development. At present, there are two main methods for producing ethanol in industry: fermentation (microorganisms use glucose to produce ethanol through anaerobic fermentation of EMP pathway or ED pathway) and chemical synthesis (using ethylene and water to directly react to produce ethanol under heating, pressure and catalyst conditions). Compared with the raw materials of chemical synthesis, the raw materials of fermentation mainly come from agricultural products such as corn, sorghum, cassava, and sugarcane, so they are more environmentally friendly and renewable. However, using grain as raw material will lead to a large amount of grain entering industrial applications, reducing the supply of grain market and causing food crisis. In order to avoid the embarrassing situation of competing with people for food, the current trend is to use cheap and abundant non-food raw materials in agricultural and forestry biomass, such as lignocellulose raw materials in agricultural residues, food and industrial waste, for industrial production of fuel ethanol.

[0003] Lignocellulosic raw materials need to be decomposed into pentose or hexose that can be used by microbial fermentation through pretreatment and saccharification steps, and then the microorganisms convert pentose or hexose into bioethanol through the EMP pathway or ED pathway. The simultaneous saccharification and fermentation is the first choice for ethanol production. Its advantages are that hydrolysis and fermentation can be carried out in a single fermentation tank, which is simple to operate, low cost, and short completion time. However, the disadvantages are that the temperature requirements between saccharification and fermentation are different. The hydrolysis step usually requires high temperature and the microorganisms are in various stress environments such as formic acid, acetic acid, furfural, etc. during the hydrolysis process, which can lead to excessive accumulation of reactive oxygen (i.e., highly active free radicals such as hydrogen peroxide, superoxide anion, hydroxyl free radicals) causing cell redox imbalance, causing serious damage to various cell components such as DNA, proteins, membrane lipids and cytoskeleton, and even leading to cell death, affecting fermentation efficiency. Therefore, anti-oxidative stress is one of the important properties that microorganisms must have in industrial fermentation. It is very necessary to separate and screen fermentation strains with excellent anti-oxidative stress properties from nature to improve the industrial fermentation efficiency of microorganisms and reduce production costs.

[0004] Currently, the ethanol production strain commonly used in industry is Saccharomyces cerevisiae. However, Saccharomyces cerevisiae has poor antioxidant properties, which makes it unable to grow well and produce ethanol under high temperature and other stress environments (such as formic acid, acetic acid, ethanol, furfural, etc.).

[0005] In summary, based on the production needs of environmentally friendly bioethanol, screening non-traditional yeast strains with excellent antioxidant stress performance is an important way to solve the problem of industrial yeast strains' intolerance to environmental stress. [Summary of the invention]

[0006] In view of this, the object of the present invention is to provide a non-traditional yeast strain with excellent antioxidant properties and use it in bioethanol fermentation.

[0007] To achieve the above-mentioned purpose, the present invention screened and obtained a Candida nivariensis GXAS-CN strain, whose preservation number is GDMCC No.62755, the preservation date is September 1, 2022, the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, and the preservation unit is Guangdong Microbiological Culture Collection Center (GDMCC).

[0008] The Candida nivariensis GXAS-CN strain of the present invention is derived from a rotten fruit sample. The strain obtained by screening is subjected to genome extraction, PCR amplification, and ITS sequence amplification to obtain an ITS sequence with a length of 760 bp. Homologous sequence comparison analysis shows that the ITS sequence of the strain has a homology of 100% with that of Candida nivariensis, and the strain is classified as Candida. The ITS sequence of the Candida nivariensis GXAS-CN strain is shown in the sequence table.

[0009] In the present invention, it is further described that the Candida nivariensis GXAS-CN strain is cultured at 37°C for 10 hours to obtain an OD 600 The absorption value is above 1.29.

[0010] In the present invention, it is further described that the Candida nivariensis GXAS-CN strain can tolerate 35 mM hydrogen peroxide.

[0011] In the present invention, it is further described that the Candida nivariensis GXAS-CN strain tolerates 10 mM vanillin, 80 mM acetic acid, 35 mM formic acid, 25 mM furfural and 10% ethanol.

[0012] The present invention also provides the use of Candida nivariensis GXAS-CN strain in fermentation.

[0013] The present invention also provides a fermentation preparation method of Candida nivariensis GXAS-CN strain, which comprises the following steps: culturing the Candida nivariensis GXAS-CN strain described above, and measuring the ethanol production rate after fermentation for different time periods.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0015] The present invention isolates a Candida nivariana GXAS-CN strain with excellent antioxidant performance from a rotten fruit sample. The strain can grow well at 37°C and 42°C, and can tolerate high concentrations of hydrogen peroxide and other stress inhibitors, such as 10mM vanillin, 80mM acetic acid, 35mM formic acid, 25mM furfural and 10% ethanol. The strain can produce ethanol with concentrations of 8.1% and 10.4% by fermenting rice bran at 30°C for 48 hours and 72 hours; and can produce ethanol with concentrations of 4.6% and 5.0% by fermenting glucose at 37°C and adding 20mM hydrogen peroxide for 48 hours and 72 hours. In summary, the GXAS-CN strain of the present invention has broad application prospects in the production of environmentally friendly bioethanol.

Brief Description of the Drawings

[0016] Figure 1 The evolutionary tree constructed with the ITS sequence of Candida nivariegata GXAS-CN strain is shown;

[0017] Figure 2 Shows the growth of Candida nivariegata GXAS-CN strain at different temperatures;

[0018] Figure 3 Shows the growth curve of Candida nivariegata GXAS-CN strain under liquid culture conditions of 30°C, 37°C, and 40°C;

[0019] Figure 4 Shows the growth of Candida nivariegata GXAS-CN strain on YPD plates with different concentrations of hydrogen peroxide;

[0020] Figure 5 The cell survival rate of Candida nivariegata GXAS-CN strain after being treated with hydrogen peroxide at different concentrations for 3 hours (Figure A); the control is the cell survival rate of Saccharomyces cerevisiae after being treated with hydrogen peroxide at different concentrations for 3 hours (Figure B);

[0021] Figure 6 The tolerance of Candida nivariana GXAS-CN strain to different stress inhibitors (vanillin, acetic acid, formic acid, furfural, and ethanol) is shown;

[0022] Figure 7 The ethanol production of Candida nivariegata GXAS-CN strain using rice bran as raw material at 30°C for 24 h, 48 h, and 72 h is shown.

[0023] Figure 8 The ethanol production of Candida nivariegata GXAS-CN strain using glucose as raw material at 37°C for 24 hours, 48 ​​hours, and 72 hours is shown. [Specific implementation method]

[0024] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0025] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0026] Example 1: Screening and identification of Candida nivariensis GXAS-CN strain

[0027] 1.1 Sample collection

[0028] The rotten fruit samples were collected, sealed in sterile bags and stored at 4°C for later use.

[0029] 1.2 Isolation of strains

[0030] First, the collected sample was placed in 100 mL of sterile water and shaken for 1 hour. A 1 mL sterile pipette was used to draw 1 mL of the solution and inoculated into 100 mL of enriched medium YPD (containing 10% glucose, 2% peptone, 1% yeast extract, 100 μg / mL streptomycin sulfate), and shaken in a 37°C constant temperature shaker for 24 hours. Then, a sterile pipette was used to draw 100 μL of the culture from the enriched medium that had been cultured for 24 hours, and after gradient dilution, it was evenly spread on a separation plate containing 2% glucose, 2% peptone, and 2% agar, and cultured at 37°C for 24 hours. Next, a sterile toothpick was used to pick up a single colony grown on the plate, and a streak culture was performed on the YPD medium. After the isolated strain grew a single colony, the strain that grew a single colony was transferred to a test tube slant for storage.

[0031] The isolated strains were inoculated into 10 mL of YPD liquid culture medium and cultured at 37°C with shaking for 24 hours, and then the antioxidant capacity and ethanol production capacity of the strains were tested. The strains with good antioxidant stress resistance and high ethanol production were identified.

[0032] 1.3 Extraction of strain DNA

[0033] Inoculate a single colony of the strain to be identified in 10 mL of YPD liquid medium and culture for 24 hours, centrifuge at 12000 rpm for 10 minutes, discard the supernatant, resuspend the bacteria with sterile water, centrifuge at 12000 rpm for 10 minutes, and discard the supernatant. Use 0.5 mL of buffer (20 mM Tris-HCl, pH 7.5, 1.2 M sorbitol, 10 mM EDTA) to resuspend the bacteria and add steel beads, grind on a grinder at 59 Hz for 2 minutes, then add 0.5 mL of phenol-chloroform isoamyl alcohol, shake well and mix well, centrifuge at 12000 rpm for 10 minutes to separate the layers. Transfer the transparent aqueous phase to a new test tube, add an equal volume of phenol-chloroform isoamyl alcohol, shake well and mix well, centrifuge at 12000 rpm for 10 minutes. Transfer the transparent aqueous phase in the supernatant to a clean tube and add 2 times the volume of anhydrous ethanol. After precipitation at -20°C for 2 hours, the mixture was centrifuged at 12,000 rpm for 10 minutes, the supernatant was discarded, and the mixture was washed twice with 70% ethanol. The DNA was then air-dried at room temperature.

[0034] 1.4 Molecular identification of strains

[0035] Determine the concentration of the extracted DNA and dilute it to a suitable concentration for polymerase chain reaction (PCR).Use the universal primers ITS1 / ITS4 of the fungal ribosomal rDNA region for PCR amplification.

[0036] ITS1:TCCGTAGGTGAACCTGCGG,

[0037] ITS4:TCCTCCGCTTATTGATATGC.

[0038] The amplified products were separated by 1% agarose gel electrophoresis and recovered using a DNA purification kit and sequenced. The ITS sequence was used to construct an evolutionary tree. The results are as follows Figure 1 As shown, the strain was named Candida nivariensis GXAS-CN after analysis.

[0039] The identified Candida nivariensis GXAS-CN strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 1, 2022, with the deposit number GDMCC No.62755, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China.

[0040] Example 2: Tolerance detection of Candida nivariensis GXAS-CN

[0041] Growth of Candida nivariana GXAS-CN strain at different temperatures: Candida nivariana GXAS-CN strain was inoculated into 10 mL of YPD liquid medium and cultured for 12 hours to prepare a bacterial suspension with an OD600 of 1, which was then diluted to 10 0 , 10 -1 , 10 -2 , 10 -3 After four concentrations, 3 μL was inoculated on YPD plates and cultured at 30°C, 37°C, 40°C, and 42°C for 2 days. The results are as follows Figure 2 As shown, the Candida nivariegata GXAS-CN strain grew well at 30°C, 37°C, 40°C, and 42°C.

[0042] Growth curves of Candida nivariana GXAS-CN strain at different temperatures: Candida nivariana GXAS-CN strain was inoculated into 10 mL of YPD liquid culture medium and cultured for 12 hours to prepare a bacterial suspension with an OD600 of 1, which was then transferred to fresh YPD liquid culture medium at a 1% inoculum amount and cultured at 30°C, 37°C, and 40°C. The OD600 absorption value of the culture was detected every 1 hour until the OD600 absorption value stabilized and the detection was stopped. The growth curve was drawn according to the measurement results, such as Figure 3 As shown, the Candida nivariegata GXAS-CN strain still grows well at a high temperature of 37°C.

[0043] Candida nivariana GXAS-CN strain is tolerant to hydrogen peroxide: Candida nivariana GXAS-CN strain was inoculated into 10 mL of YPD liquid medium and cultured for 12 hours to prepare a bacterial suspension with an OD600 of 1, and then diluted to 10 0 , 10 -1 , 10 -2 , 10 -3 After four concentrations, 3 μL was inoculated on YPD plates containing different concentrations of hydrogen peroxide and cultured at 37°C for 2 days. Figure 4 As shown, the Candida nivariegata GXAS-CN strain still grew well on the YPD plate containing 35 mM hydrogen peroxide.

[0044] Comparison of the survival rate of Candida nivariana GXAS-CN strain and Saccharomyces cerevisiae after 3 hours of hydrogen peroxide treatment: Candida nivariana GXAS-CN strain and Saccharomyces cerevisiae BJ5464 strain were inoculated into 10 mL of YPD liquid culture medium and cultured for 12 hours, and 0.5 OD of bacterial cells were collected and 500 μL of YPD liquid culture medium containing different concentrations of hydrogen peroxide was added to resuspend the cells, and cultured at 30 ° C / 200 rpm for 3 hours, centrifuged to remove the culture medium, added 500 μL of sterile water to resuspend the cells, centrifuged to remove the supernatant, added 500 μL of sterile water to resuspend the cells, and the suspension was diluted to 10 0 , 10 -1 , 10 -2 , 10 -3 After four concentrations, 3 μL was inoculated onto a YPD plate and cultured at 30°C for 2 days. Figure 5 As shown, the Candida nivariegata GXAS-CN strain still had a high survival rate after being treated with 400 mM hydrogen peroxide for 3 hours ( Figure 5 A), but almost all of the Saccharomyces cerevisiae died after being treated with 20 mM hydrogen peroxide for 3 hours ( Figure 5 B), the antioxidant capacity of Candida nivariegata GXAS-CN strain was significantly better than that of Saccharomyces cerevisiae.

[0045] Tolerance of Candida nivariana GXAS-CN strain to different stress inhibitors: Candida nivariana GXAS-CN strain was inoculated into 10 mL of YPD liquid medium and cultured for 12 h to prepare a bacterial suspension with an OD600 of 1, which was then diluted to 10 0 , 10 -1 , 10 -2 , 10 -3 After four concentrations, 3 μL was inoculated on YPD plates containing different concentrations of stress inhibitors and cultured at 37°C for 2 days. Figure 6 As shown, the Candida nivariegata GXAS-CN strain tolerated 10 mM vanillin, 80 mM acetic acid, 35 mM formic acid, 25 mM furfural, and 10% ethanol.

[0046] Example 3: Ethanol production of Candida nivariensis GXAS-CN strain

[0047] The ethanol production of Candida nivariegata GXAS-CN strain using rice bran as raw material at 30℃ for 24 hours, 48 ​​hours, and 72 hours. Figure 7 As shown, the ethanol production of the strain of the present invention can reach 8.1% and 10.4% after culturing for 48 hours and 72 hours.

[0048] The ethanol production of Candida nivariegata GXAS-CN strain was measured by fermentation with 100 g / L glucose at 37°C and 20 mM hydrogen peroxide for 24, 48, and 72 hours. Figure 8 As shown, the ethanol production of the strain of the present invention can reach 4.6% and 5.0% after culturing for 48 hours and 72 hours.

[0049] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Candida nivariegata ( Candida nivariensis ) GXAS-CN strain, its deposit number is GDMCC No.62755, the deposit date is September 1, 2022, the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, and the deposit unit is Guangdong Microbiological Culture Collection Center (GDMCC).

2. The Candida nivariegata according to claim 1 ( Candida nivariensis ) Application of GXAS-CN strain in ethanol fermentation.

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