Paecilomyces variotii and application thereof

Abstract

The invention discloses paecilomyces variotii PYJ1, the classification name of the paecilomyces variotii PYJ1 is paecilomyces variotii PYJ1, the paecilomyces variotii PYJ1 is preserved in the China Center for Type Culture Collection on March 6, 2024, and the preservation number of the paecilomyces variotii PYJ1 is CCTCC (China Center for Type Culture Collection) NO: M2024348. The Paecilomyces variotii PYJ1 can completely degrade main inhibitors including acetic acid, furfural, 5-hydroxymethylfurfural and the like generated when corn straw is pretreated with sulfuric acid within 48 hours, detoxification is carried out under the mild conditions of room temperature, no ventilation and no stirring, and the defects that a traditional biological detoxification method is long in period, incomplete in inhibitor degradation, large in energy consumption and the like are overcome; and the straws subjected to biological detoxification can be used as edible fungus cultivation raw materials or can be subjected to subsequent enzymolysis and biological conversion.

Description

[Technical field]

[0001] The invention belongs to the field of microbial engineering, and in particular relates to a strain of Paecilomyces variotii and the degradation of inhibitors produced in a process of sulfuric acid treatment of corn stalks by the strain. [Background technology]

[0002] In the agricultural production process, after harvesting corn, wheat, rice and other food crops, the remaining inedible stalks, stems, leaves and other byproducts are called crop straw. Crop straw is a major biomass resource, but in recent years, with the rapid development of the economy and the improvement of the energy structure, many regions have experienced seasonal and structural straw surpluses, which are specifically manifested in the piles of abandoned crop straw and burning on the spot. The random burning of straw not only pollutes the natural ecological environment, but also causes a waste of resources. Especially in the context of the increasing scarcity of non-renewable resources such as fossil energy, the recyclable and high-quality development of crop straw has a broader application prospect.

[0003] Crop straw is a typical lignocellulose and a renewable resource. The development and application of lignocellulose has been a hot topic in recent years. Edible fungi are high-value products, and the use of straw as a cultivation medium for edible fungi has attracted widespread attention. Straw has the characteristics of loose structure, good air permeability, and strong water retention. Using artificial cultivation methods to process discarded crop straw into a substrate for the growth of edible fungi can greatly improve the utilization efficiency of straw and reduce the production cost of edible fungi. The hyphae of edible fungi will secrete an enzyme mixture to degrade lignocellulose, and eventually decompose it into monosaccharides that can be used for their own growth and metabolism, realizing the transformation of crude fiber into edible fungi that can be eaten by humans.

[0004] Due to the complex structure of lignocellulose, it must be pretreated before specific applications. The most commonly used method is acid pretreatment. Pretreatment can destroy the cell wall structure of lignocellulose, making cellulose easier to be hydrolyzed by enzymes and converted into bioavailable sugars. In this process, some biologically toxic substances, inhibitors (including acetic acid, furfural, 5-hydroxymethylfurfural, etc.), will inevitably be produced. How to effectively remove inhibitors has a great impact on the subsequent fiber saccharification, microbial fermentation, and the quality of the substrate used as edible fungus cultivation. In recent years, the biological detoxification method using microorganisms to degrade inhibitors has received increasing attention. CN102191279A discloses a strain of Amorphotheca resinae ZN1 that can degrade inhibitors such as furfural, but the degradation of the inhibitor usually takes 3 to 6 days; CN105734087A discloses a strain of Amorphothecaresinae ZN1 that can partially degrade inhibitors in 36 hours, but the detoxification process requires ventilation and intermittent mechanical stirring, and the degradation of the inhibitor is not complete (furfural is about 70%, 5-hydroxymethylfurfural is about 40%, and acetic acid is only degraded by 3%). In view of the fact that the current biological detoxification method generally has a long cycle (5 to 10 days), the degradation of the inhibitor is incomplete, or ventilation or stirring is required, resulting in a significant increase in the energy consumption of biological detoxification, and the detoxification efficiency and cost remain high, screening microorganisms that can economically and efficiently degrade lignocellulose-derived inhibitors is of great significance for the high-value utilization of agricultural and forestry wastes.

[0005] The present invention screens the target microorganisms through two stages: soil enrichment and adaptive evolution. First, 11 strains of microorganisms that can tolerate acetic acid, furfural and 5-hydroxymethylfurfural were preliminarily screened from soil samples in East China University of Science and Technology, and then the target microorganism (Paecilomyces veriotii PYJ1) with growth advantage and a degradation rate of the main inhibitor within 48 hours of more than 90% was obtained by means of adaptive evolution. The microorganism was used to achieve efficient degradation of inhibitors in straw after acid pretreatment. [Summary of the invention]

[0006] In order to solve the above-mentioned shortcomings of the prior art, one of the objects of the present invention is to provide a Paecilomyces veriotii PYJ1, which has been preserved by the China Center for Type Culture Collection with a preservation number of CCTCC M2024348 and a preservation date of February 28, 2024.

[0007] The colony diameter of the screened strain PYJ1 after culturing on a PDA plate at 30°C for 4 days was 2-3 cm; the colony was white at the beginning, and became light yellow and fluffy after spore production, with a bulge in the middle and a dark yellow reverse side; under an optical microscope, the hyphae were relatively thick, colorless and transparent, and had septa; the carbon sources that PYJ1 could utilize included one or more of glucose, xylose, arabinose, galactose, and mannose; the growth pH range was 5.0-6.0; the most suitable growth temperature was 25-30°C; the ITS1-ITS4 sequence of PYJ1 (SEQ ID 1) was used for sequence alignment and a phylogenetic tree was constructed, and the phylogenetic tree results are shown in the attached Figure 1 , PYJ1 and Paecilomyces veriotii were classified into the same species; strain PYJ1 was identified as Paecilomyces veriotii.

[0008] Another object of the present invention is to provide the use of the above-mentioned Paecilomyces variotii to degrade the main inhibitors produced in the process of sulfuric acid treatment of corn stalks. The fungus can grow in the stalks pretreated with dilute sulfuric acid and degrade the inhibitors therein, including one or more of furfural, 5-hydroxymethylfurfural, acetic acid, syringaldehyde and vanillin.

[0009] Compared with the prior art, the present invention has the following positive effects:

[0010] The biological detoxification cycle is shorter, and the inhibitors produced in the pretreatment can be completely degraded within 48 hours; the inhibitors are completely degraded, and the concentrations of major inhibitors such as acetic acid, furfural and 5-hydroxymethylfurfural, formic acid, acetic acid, syringaldehyde and vanillin in the straw after biological detoxification are less than 0.02g / L; the biological detoxification operation is simple, and no ventilation and stirring are required, and the inhibitors can be degraded at room temperature.

[0011]

Attached chart description

[0012] Figure 1 Phylogenetic tree of strain PYJ1. [Specific implementation method]

[0013] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.

[0014] Example 1 Main inhibitors in straw after sulfuric acid pretreatment

[0015] Before sulfuric acid pretreatment, corn stalks were first crushed with a crusher to a particle size between 20 and 80 meshes. The sulfuric acid pretreatment process was as follows: 200g of crushed corn stalks were weighed in a packaging bag, 5% sulfuric acid was added, stirred evenly and allowed to stand for 3 hours to allow it to fully react, and then wet heat acid treatment was performed at 160°C for 180 minutes. After taking it out, calcium oxide was added for neutralization and the pH was adjusted to neutral. The moisture content of the pretreated stalks was 53%.

[0016] The inhibitors in the pretreated straw were determined by high performance liquid chromatography. 5g of pretreated straw was dissolved in 45g of pure water, placed in a shaker at 220rpm and 30℃ and mixed for 30min, then taken out and poured into a 50ml centrifuge tube, placed in a centrifuge at 7500rpm and centrifuged for 30min, and the supernatant was taken and filtered through a membrane for liquid phase determination. The concentration of furfural was 0.25g / L, the concentration of 5-hydroxymethylfurfural was 0.41g / L, and the concentration of acetic acid was 0.15g / L.

[0017] Example 2 Biodetoxification of corn stover treated with sulfuric acid by Paecilomyces veriotii PYJ1

[0018] Elute the spores from the tube slant with sterile water and dilute accordingly to obtain 10 6 ~10 7 Spore suspension. Inoculate into pretreated straw with a moisture content of 53% at an inoculation rate of 10%, adjust the humidity to about 70%, place in a 30°C constant temperature box and culture until the straw is covered with mycelium to serve as seed straw for subsequent experiments. Inoculate PYJ1 seed straw into 5g of corn straw pretreated in Example 1 at an inoculation rate of 10%, add 3ml of sterile water to keep its humidity at about 70%, place in a 30°C incubator for solid detoxification, and prepare corresponding duplicate samples according to the needs of detection and experiments. Turn the material every 24h, take samples and measure. During the measurement, take a duplicate sample, add 45ml of pure water, put it in a 220rpm, 30°C shaker and mix for 30min, then take it out and pour it into a 50ml centrifuge tube, put it in a 7500rpm centrifuge for 30min, take the supernatant, and perform liquid phase analysis on the inhibitor. The results are shown in Table 1. Furfural and acetic acid were completely degraded within 24 hours, and 5-hydroxymethylfurfural was completely degraded within 48 hours. It can be seen that Paecilomyces veriotii PYJ1 has a significant detoxification effect on inhibitors.

[0019] Table 1 Biodetoxification of pretreated straw by Paecilomyces veriotii PYJ1

[0020]

[0021] Example 3 Biodetoxification of corn stover treated with sulfuric acid and supplemented with inhibitors by Paecilomyces veriotii PYJ1

[0022] As can be seen from Example 2, Paecilomyces veriotii PYJ1 has a very impressive ability to degrade inhibitors in pretreated straw. Therefore, Example 3 conducted an experiment to add additional inhibitors to solid straw to further explore its degradation ability. According to the method of Example 2, inoculation and preparation of corresponding replicates were performed, and 0.05g and 0.1g of inhibitors were added to 5g of corn straw pretreated in Example 1, respectively, so that the initial concentrations were about 1g / L and 2g / L respectively when liquid phase sampling was performed (the sampling and liquid phase determination methods are shown in Example 2), and placed in a 30°C constant temperature incubator for detoxification treatment. The experimental results are shown in Table 2. Paecilomyces veriotii PYJ1 completed the detoxification of 1g / L and 2g / L furfural within 3 days and 5 days, respectively; 1g / L and 2g / L 5-hydroxymethylfurfural were detoxified within 5 days and 7 days, respectively; 1g / L and 2g / L acetic acid were detoxified within 2 days and 3 days, respectively.

[0023] Table 2 Biodetoxification of pretreated straw with additional inhibitors by Paecilomyces veriotii PYJ1

[0024]

[0025] Example 4 Testing of using biologically detoxified straw as a substrate for growing edible fungi

[0026] In order to verify the actual detoxification effect of Paecilomyces veriotii PYJ1, an edible fungus cultivation experiment was carried out using the detoxified straw. Example 1 The moisture content of the straw after pretreatment was 53%. Sterile water was added to the straw to adjust the moisture content to 70%. The detoxification experiment used non-detoxified straw as the control group. The detoxification bag was placed in an environment with an ambient temperature of 23-26°C and a humidity of 60%-70% for detoxification. The detoxification bag was opened and rolled inward to avoid contamination by miscellaneous bacteria. Samples were taken every day to detect the inhibitor content until the inhibitor content could not be detected, and the detoxification was stopped.

[0027] The detoxified straw was mixed with the purchased edible fungus bag substrate in a ratio of 1:1 and placed in an environment with a temperature of 25°C and a humidity of 70% for a fruiting experiment. After the mycelium in the original fungus bag was fermented in the newly configured fungus bag, its mycelium grew all over the fungus bag again and entered the fruiting stage. When the oyster mushroom is too mature, a large number of spores will be produced on its gills. These spores will be scattered around and released into the air, resulting in a decrease in the fruiting yield. Therefore, the best time to pick is when the cap is expanded to the maximum and before a large number of spores are produced. The experimental results are shown in Table 3. The untreated straw group fruited on the 13th day with a fruiting amount of 40.3g. The detoxified straw of Paecilomyces veriotii PYJ1 fruited on the 8th day with a fruiting amount of 85.9g. From the results, it can be seen that the new fungus bag configured with biologically detoxified straw can be used to replace the traditional cultivation substrate to complete the fruiting and fruiting of edible fungi.

[0028] Table 3 Mushroom yield

[0029]

Claims

1. A strain of Paecilomyces variotii and its use, characterized in that: The strain is Paecilomyces veriotus PYJ1, which was deposited in the China Center for Type Culture Collection on February 28, 2024, with the deposit number CCTCC NO: M2024348.

2. According to claim 1, it is characterized in that Paecilomyces variotii PYJ1 can degrade the main inhibitor produced when corn stover is pretreated with sulfuric acid.

3. According to claim 2, it is characterized in that The main inhibitors produced by the sulfuric acid pretreatment of corn stover include, but are not limited to, one or more of acetic acid, furfural and 5-hydroxymethylfurfural.

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