Eucalyptus cellulose degrading bacterium QS2 and application thereof

Abstract

The invention relates to the technical field of eucalyptus degradation, in particular to eucalyptus cellulose degrading bacteria QS2 and application thereof. The classification name of the strain is Aspergillus fumigaster, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 3, 2024, the preservation number is CGMCC No: 41393, and the strain has important significance for degrading eucalyptus residues and improving the soil fertility of eucalyptus artificial forest land.

Description

Technical Field

[0001] The present application relates to the technical field of eucalyptus degradation, and in particular to a eucalyptus cellulose degrading bacterium QS2 and its application. Background Art

[0002] Eucalyptus is a high-quality fast-growing tree species in tropical and subtropical regions. It is a short-cycle fiber material with good economic benefits. With the support of industrial policies, the planting area of ​​eucalyptus has gradually increased. However, with the continuous planting of eucalyptus for many generations and the continuous expansion of planting area, ecological problems have also emerged, such as decreased soil fertility, reduced microbial diversity and number, severe soil erosion, and reduced forest productivity.

[0003] The supply of soil organic matter is one of the key factors that cause the decline of soil fertility in eucalyptus plantations. In related technologies, the model of non-burning afforestation is often adopted, that is, retaining logging residues on felled land. Compared with burning the residues, retaining the residues can replenish a large amount of nutrients in the soil under the forest, which is an important source of forest soil organic matter and the basis for maintaining forest soil fertility.

[0004] However, simply retaining the residues will result in a slow decomposition rate and easy fire. The higher the decomposition rate of the residues, the more humus they produce, which can effectively improve soil quality, improve the physical structure of the soil, and increase the nutrients in the soil. The main component of eucalyptus residues is cellulose, and the degradation of cellulose mainly depends on cellulose-degrading bacteria. However, there is little research on eucalyptus cellulose-degrading bacteria. Therefore, in view of the need to degrade eucalyptus felling residues as quickly as possible, it is of great significance to study how to promote the degradation of eucalyptus residues. Summary of the invention

[0005] The present application provides a eucalyptus cellulose-degrading bacterium QS2 and its application to solve the problem of lack of eucalyptus cellulose-degrading bacteria in the related art.

[0006] In the first aspect, a eucalyptus cellulose-degrading bacterium QS2 is provided, which is classified and named Aspergillus fumigatus, and was deposited in the General Microbiology Center of China Culture Collection Committee on July 3, 2024, with a deposit number of CGMCC No: 41393.

[0007] In a second aspect, a use of the eucalyptus cellulose degrading bacteria QS2 as described above in eucalyptus degradation is provided.

[0008] The beneficial effects of the technical solution provided by this application include:

[0009] The embodiment of the present application provides a eucalyptus cellulose-degrading bacterium QS2 and its application. By isolating and purifying eucalyptus soil samples, a QS2 strain with high enzyme activity is screened out, which has significant advantages in the degradation of eucalyptus residues, improves the conversion and utilization rate of cellulose substances, and thus increases the soil fertility of eucalyptus plantations. Therefore, it can solve the problem of lack of eucalyptus cellulose-degrading bacteria in related technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0011] Figure 1 A schematic diagram of the transparent circle of strain QS2 provided in this application;

[0012] Figure 2 This is a morphological observation diagram of strain QS2 provided in this application;

[0013] Figure 3 A schematic diagram of strain QS2 provided for this application;

[0014] Figure 4 A schematic diagram of the filter paper disintegration of strain QS2 provided in this application on the 3rd day;

[0015] Figure 5 A schematic diagram of the filter paper disintegration of strain QS2 provided in this application on day 5;

[0016] Figure 6 Schematic diagram of filter paper disintegration of strain QS2 provided in this application on the 7th day. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0018] See also Figures 1 to 6 As shown, the present application provides a eucalyptus cellulose degrading bacterium QS2, which can solve the problem of lack of eucalyptus cellulose degrading bacteria in the related art.

[0019] Eucalyptus soil samples and rotten branches and leaves were collected in Liuzhou and Nanning, the main eucalyptus producing areas in Guangxi.

[0020] The cellulose-degrading fungi in the selected soil samples were preliminarily screened.

[0021] Add 10 mL of sterile water to the conical flask, weigh 1 g of the eucalyptus soil sample and add it to the conical flask, then put the conical flask into a constant temperature shaker at a constant temperature of 28°C for 30 minutes to fully mix the soil sample and sterile water to obtain a soil homogenate;

[0022] The soil solution was diluted to 10 -1 , 10 -2 , 10 -3 Use a pipette to absorb 100 μL of soil suspension of each concentration, and evenly apply it to the cooled and solidified cellulose screening medium. After the soil suspension penetrates into the cellulose screening medium, seal it with a sealing film and then place it in a constant temperature incubator at 28°C for 3 to 5 days.

[0023] Twenty strains were isolated and purified, and one strain with an obvious transparent zone was re-inoculated into sterilized eucalyptus thick branches and cultured on thin branches. After mycelium grew out, the mycelium was picked up and placed in a culture dish, namely QS2.

[0024] In some embodiments, the cellulose screening medium includes 5.0 g sodium carboxymethyl cellulose, 3.0 g sodium nitrate, 1.0 g potassium dihydrogen phosphate, 0.6 g magnesium sulfate heptahydrate, 0.5 g potassium chloride, 0.001 g ferrous sulfate heptahydrate, 15.0 g agar, and 1000 mL distilled water.

[0025] Characterization Tests:

[0026] After the culture is completed, the growth of cellulose-degrading microorganisms cultured on the cellulose culture medium is observed and recorded. Through this experiment, the strain QS2 with high cellulose degradation ability was screened. A single bacterium was inoculated onto the Congo red culture medium with a sterilized pipette tip, and cultured at 28°C for 4 days. The transparent circle around the colony was observed; the ratio of the transparent circle diameter to the colony diameter was used as the standard to preliminarily judge the cellulase production capacity of each colony. The ratio of the transparent circle diameter to the colony diameter after 4 days of culture was used as the screening standard. The experimental results are as follows: Figure 1 and Table 1. Its colony shape is irregular circle and its colony color is green. Observed under an optical microscope, see Figure 1 and Figure 2 As shown, QS2 has an irregular circular shape, and the spore-producing area is located in the middle of the colony, with conidia in the shape of concentric circles and green color. There are obvious upright hyphae.

[0027] Table 1

[0028]

[0029]

[0030] See also Figure 1 As shown, QS2 has an obvious transparent zone, and the diameter of the transparent zone / colony is 2.08.

[0031] Filter paper disintegration test:

[0032] The strain was inoculated into the filter paper liquid culture medium. Under the conditions of 28°C and 180r / min, the filter paper was taken out on the 3rd, 5th and 7th day of culture to measure the weight loss rate. The filter paper was fully dried in an oven at 100°C before and after disintegration. The weight loss rate (X) of the filter paper was used as the standard, and the formula was:

[0033] X=(m-m1) / m

[0034] Where m is the initial weight of the filter paper, and m1 is the weight of the filter paper after degradation.

[0035] The strains screened by Congo red plate were cultured in filter paper liquid culture medium for different time periods, and the filter paper weight loss results are shown in Table 2.

[0036] Table 2

[0037] strain Weight loss rate after 3 days Weight loss rate after 5 days Weight loss rate after 7 days QS2 41.70％ 45.02％ 28.83％

[0038] See also Figure 3 to Figure 6 As shown, Figure 4 , 5 6 are schematic diagrams of filter paper disintegration on the 3rd, 5th, and 7th days, respectively. After 1 day, the colonies gradually attached to the filter paper and grew, and the filter paper was basically degraded; after the 3rd day, the filter paper was completely degraded, and the culture medium was green. Other strains can basically be completely degraded after the seventh day. The colony shape is irregular circle, and the colony color is green.

[0039] Enzyme activity test:

[0040] The bacteria obtained from the initial screening were inoculated into the seed culture medium and cultured at 28°C, 180r / min for 3 days to obtain uniform seeds, and then inoculated into the liquid enzyme production identification culture medium at a ratio of 5% and cultured at 28°C, 180r / min. After culturing for 4 days, the crude enzyme was obtained by centrifugation, and its FPA enzyme activity and CMC enzyme activity were determined respectively.

[0041] Among them, the calculation formula of FPA enzyme activity is:

[0042] X1=A×1 / 0.5×n

[0043] Where, X1—filter paper enzyme activity (FPA) of the sample, u / g (or u / mL);

[0044] A—reducing sugar amount found (or calculated) on the standard curve according to absorbance, mg;

[0045] 1 / 0.5—converted into 1mL of enzyme solution;

[0046] n—dilution factor of enzyme sample.

[0047] CMC enzyme activity calculation formula:

[0048] X1=A×1 / 0.5×n×2

[0049] Where, X1 is the carboxymethyl cellulase activity (CMCA-DNS) of the sample, u / g (or u / mL); A is the amount of reducing sugar found (or calculated) on the standard curve according to the absorbance, mg;

[0050] 1 / 0.5—converted into 1mL of enzyme solution;

[0051] n—dilution multiple of enzyme sample;

[0052] 2—Time conversion factor.

[0053] The results of FPA and CMC enzyme activities are shown in Table 3.

[0054] Table 3

[0055] strain FPA CMC QS2 1.98 7.01

[0056] In summary, the filter paper disintegration rate of QS2 after 3 days was 41.7%, the FPA enzyme activity was 1.98, and the CMC enzyme activity was 7.01. Its enzyme activity was high, which could degrade eucalyptus residues well, thereby increasing the soil fertility of eucalyptus plantations, and playing a good role in the degradation of eucalyptus residues.

[0057] The gene sequence of the eucalyptus cellulose-degrading bacterium QS2 (SEQ ID No. 1) is:

[0058] .

[0059] And the application of eucalyptus cellulose degrading bacteria QS2 in eucalyptus degradation:

[0060] The following is the determination of the degradation rate of eucalyptus:

[0061] The eucalyptus residue was dried at 60°C to constant weight, crushed and sieved (40 mesh), and each triangular bottle was filled with 90mL of liquid culture medium with eucalyptus powder as the sole carbon source, where 2g of eucalyptus powder was added to each bottle, and sterilized at 121°C for 20min; each bottle was inoculated with 10mL of QS2 bacterial suspension, cultured on a shaker at 28°C for 20d, and centrifuged at 5000r / min for 10min, the supernatant was discarded, the residue was repeatedly centrifuged and washed 3 times with distilled water, and then dried at 60°C to constant weight, and the weight loss rate was calculated. The control group was eucalyptus liquid culture medium inoculated with 10mL of sterile water, and 3 repeated experiments were set up.

[0062] The weight loss rate formula is X = (m-m1) / m, where X is the weight loss rate (degradation rate), m is the initial weight of eucalyptus powder, and m1 is the weight of eucalyptus powder after degradation. Through the determination of the degradation rate of eucalyptus by strains, it was found that strain QS2 has a certain degradation effect on eucalyptus, and the degradation rate was determined to be 29.0%.

[0063] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A eucalyptus cellulose degrading bacterium QS2, characterized in that: Its classification name is Aspergillus fumigatus, and it was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 3, 2024, with the deposit number CGMCCNo: 41393.

2. Use of the eucalyptus cellulose degrading bacteria QS2 as claimed in claim 1 in eucalyptus degradation.

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