CN111705008A - Bacillus firmus CX8 from termite intestinal tract and application thereof - Google Patents

Abstract

The invention discloses a bacillus firmus CX8 from termite intestinal canal, which is preserved in Beijing 3 Chaoyang district Beichen Xilu No.1 institute No. 3 China microorganism strain preservation management Committee common microorganism center in 8 months and 27 days in 2018, with the preservation number: CGMCC No. 16342. The bacterial strain is gram-positive bacillus without capsule, endophytic spores can be seen by spore staining, the bacterial colony is circular and single, the color of the bacterial colony is white, and the edge of the bacterial colony is complete. The strain is a facultative anaerobic strain for producing cellulase; the CMCase enzyme activity of the strain is 67.8IU/mL within 48h, and the CX enzyme activity is 20.96 IU/mL. The strain not only has high biodiversity value, but also has scientific value of further research in the development and application of cellulose-enriched crops and traditional Chinese medicine fermentation recycling technology.

Description

Bacillus firmus CX8 from termite intestinal tract and application thereof

Technical Field

The invention relates to a bacillus firmus CX8 from termite intestinal tracts and application thereof.

Background

Lignocellulose is a main structural component in plant cell walls, and lignin, cellulose and hemicellulose 3 are mutually embedded and combined by covalent bonds to form a spatial network structure. Because lignocellulose has large molecular weight, complex structure and stable property, the lignocellulose is extremely difficult to decompose under natural conditions. Various crop straws, economic crops and medicinal plant wastes rich in lignocellulose in agricultural production in China have abundant resources and huge yield. However, due to the influence of factors such as life style, processing technology and poor feeding performance of the processing technology, a large amount of crop straws and medicinal plant wastes are discarded or incinerated for a long time and are not reasonably developed and utilized. According to investigation, the utilization rate of the straws in China is about 33 percent at present, most of the straws are not treated, and the straws are only utilized by about 2.6 percent after technical treatment. Therefore, the development of comprehensive utilization research of lignocellulose has important practical significance for saving resources, protecting the environment, increasing the income of farmers and promoting the sustainable development of agriculture. The common fiber degradation methods at present include mechanical degradation, acid degradation, alkali degradation, thermal degradation, oxidative degradation, electrochemical degradation and biological enzymolysis. Compared with other methods, the biological enzymolysis method utilizes cellulase to catalyze the cellulose degradation, and is a mild, rapid and environment-friendly method. The varieties of the organisms producing cellulase in nature are various, and the molecular size, enzyme activity, reaction conditions and expression level of cellulase genes in different microorganisms of the produced cellulase are different, so that the organism sources producing cellulase need to be screened and identified. Termites are rare wood-eating insects which can efficiently utilize lignocellulose in the nature, the special microbial environment existing in intestinal tracts of the termites is the main reason for efficient degradation of the cellulose, and particularly in tropical regions, the decomposition level of intestinal bacteria of the termites on plant cell wall polysaccharide is 30% -40% higher than that of large-scale herbivorous animals in the same region.

The termite gut is an important ecosystem that is populated by a variety of microorganisms, including bacteria, protists, fungi and archaea, since host microbial interactions are important causes of effective degradation of lignocellulose. The termite intestinal canal can be regarded as an anaerobic gradient system, oxygen is continuously supplied through epithelial cells, exchange among the colony members of the intestinal flora is transmitted to the next generation through a nutritional axis (nutritional axis), and thus, the diversity of symbiotic microorganisms and a host system is promoted to be generated during the co-evolution. Termites are complex groups of different species of termites, roughly divided into lower termites and higher termites, which have dense and diverse prokaryotes and flagellar protists in their intestinal tracts. Protozoan symbionts living in the lower layer of termites are responsible for digestion of lignocellulose, the development and composition of host-specific systems of the protozoans reflect the obligate (reciprocal) symbiotic relationship between the protozoans and the termites, higher termites lack flagella and only contain prokaryotes in the internal organs of the high structures of the higher termites, but both the higher termites and the lower termites can produce own cellulolytic enzymes. The method proves that the intestinal tracts of the termites really have the bacteria capable of degrading the cellulose. The barrier to the development of termite gut symbiotic systems is the isolation and culture of large numbers of gut microbes, and culture techniques are often unsuitable for their development given the stringent environmental conditions required for gut bacteria to survive and reproduce. These problems have been overcome by independent culture methods, which provide an effective opportunity to study the phylogenetic abundance of termite gut bacteria. In particular, based on 16s rrna gene sequencing and fingerprinting, a stable information flow was generated regarding the abundance of various termite enterobacteria species. The test aims to separate the bacterial strain capable of degrading cellulose from the intestinal tracts of the yellow thorny fin termites with low wood food and the like, and screen out the dominant bacterial strain which has high cellulose degradation capability and is easy to culture and reproduce by measuring the enzyme activity, thereby providing effective technical support for recycling crop straws, economic crops and medicinal plant wastes.

Disclosure of Invention

The invention aims to provide a bacillus firmus CX8 from termite intestinal tracts and application thereof.

A Bacillus firmus CX8 (Bacillus firmus) from termite intestinal tracts is preserved in China general microbiological culture collection center No. 3 of West Lu No.1 Hospital in Beijing 3 rising area for 27 days 8 and 8 months in 2018, and the preservation numbers are: CGMCC No. 16342.

The bacterial strain is gram-positive bacillus without capsule, endophytic spores can be seen by spore staining, the bacterial colony is circular and single, the color of the bacterial colony is white, and the edge of the bacterial colony is complete.

The strain is a facultative anaerobic strain for producing cellulase; the CMCase enzyme activity of the strain is 67.8IU/mL within 48h, and the CX enzyme activity is 20.96 IU/mL.

The bacillus firmus CX8 is developed and applied in the fermentation and reutilization technology of cellulose-enriched crops and traditional Chinese medicines.

Also can be used for the application of converting lignocellulose into bioethanol and the application of fermenting and recycling Chinese medicinal waste.

The invention takes carboxymethyl cellulose as a matrix, establishes an enrichment medium, and preliminarily screens out strains CX1-CX10 with high cellulase yield by comparing the ratio of the transparent hydrolysis circle of each bacterium to the diameter of a bacterial colony after congo red dyeing liquid is used. The cellulase can be divided into three types of exo-beta-1, 4-glucosidase, endo-beta-1, 4-glucosidase and beta-1, 4-glucolase, and the invention further screens bacteria separated and purified from the intestinal tracts of termites by measuring the enzyme activities of three enzyme systems of cellulose endo-cellulase (CMCase), filter paper enzyme activity (FPA) and cotton enzyme activity (CX) in the strains and superposing the three enzyme activities for comparison. CX8 strain which is CX8 has great advantages in cellulase production and enzyme activity is screened out. The screened termite intestinal cellulose degradation strain CX8 in the research has high biodiversity value, and the development and application in the cellulose-enriched crop and traditional Chinese medicine fermentation recycling technology have scientific value for further research.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a transparent hydrolysis loop of an isolated strain of the invention;

FIG. 2 is a glucose standard curve for determining cellulase activity;

FIG. 3 is a measurement of enzyme activity after fermentation of the strain of the present invention;

FIG. 4 is a microscopic morphology of the strain of the present invention;

FIG. 5 shows PCR amplification of the 16SrDNA gene of the strain of the invention; wherein M is 2000 bpDNAake, and 1-3 respectively represent strains CX8, CX9 and CX 10;

FIG. 6 is a homoclade of strains CX2, CX3, C4, CX5, CX8, CX9, CX 10.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

1 materials and methods

1.1 test materials

Termites were purchased from Chuzhou, Anhui and all were low-grade wood-eaten Scopolia sinensis. After the termite is purchased, the termite is cultured in a laboratory for one week, and the termite is tested after living stably.

1.1.1 Primary reagents and instruments

LB agar medium, MRS broth, blood plate culture, nutrient broth, bacterial genome DNA kit, bacterial PCR kit, gram staining solution, capsular staining solution, spore staining solution, all purchased from Beijing Solibao science and technology, Inc.; sodium carboxymethylcellulose culture medium (CMC-Na), sodium carboxymethylcellulose Congo red culture (CMC-Na Congo red culture medium), Congo red purchased from Shandong topological bioengineering Co., Ltd; the main instruments are an electronic balance JY502 (Shanghai Puchun measuring instruments Co., Ltd.), an anaerobic incubator 1029 (Thermoscientific Co., Ltd.), a biological safety cabinet HFsafe-1500 (Likang biomedical science and technology Co., Ltd.), an ultraviolet spectrophotometer EVO300LC model, a PCR instrument 9002 model and ABI company.

1.2 methods

1.2.1 Termite treatment

Sterilizing 20 termites with 75% alcohol for 5min, pulling out the whole intestinal tract of termite under test with forceps on a sterile operating platform, mixing with a homogenizer, taking 1mL homogenate liquid, and performing gradient dilution with sterile normal saline with dilution times of 10¹，10²，10³，10⁴，10⁵，10⁶。

1.2.2 preliminary screening of the Strain

Respectively take 10⁴，10⁵，10⁶100 mul of termite liquid diluent was coated on CMC-Na, LB agar and blood plate solid medium, and 3 parallel tests were performed for each dilution gradient. And respectively putting the coated solid flat plates into a 37 ℃ constant-temperature incubator and a 37 ℃ anaerobic incubator, carrying out inverted culture for 24h/72h, observing the growth condition of bacteria, selecting strains with good growth states, and further re-screening.

1.2.3 Strain rescreening

The selected pure culture colonies are streaked in a CMC-Na culture medium, cultured for 24 hours at 37 ℃, then single colonies are selected to be spotted on CMC-Na congo red solid plates, 4 strains are spotted on each plate, and 3 parallel tests are carried out. The diameter (D) of the zona pellucida and the diameter (D) of the colonies were measured, respectively, and the D/D ratio of each strain was calculated. And selecting strains with obvious transparent hydrolysis rings around the colonies for purification.

1.2.4 purification

Selecting single bacterial colony after secondary screening, dibbling the single bacterial colony on a primary screening culture medium, continuously carrying out streak culture for 3 times at 37 ℃, selecting single bacterial colony with good growth state, carrying out streak culture on a CMC-Na culture medium, carrying out Congo red staining, carrying out gram staining and microscopic examination on the bacterial strain with an obvious transparent ring, observing the staining characteristics of bacteria and the uniformity of morphological characteristics, and numbering.

1.3 determination of cellulase Activity

1.3.1 preparation of crude enzyme solution

Taking each culture strain, inoculating 8% of the strain in a triangular flask filled with 100mLCMC-Na liquid fermentation medium, filter paper culture medium and absorbent cotton ball culture medium, respectively, and performing shake culture at 37 deg.C and 120r/min in a constant temperature shaking table. Centrifuging 2mL fermentation liquid for 5min at 10000r/min after 48h to remove thallus, and collecting supernatant as crude enzyme solution^[4]。

1.3.2 drawing of glucose Standard Curve

100mL of a 1mg/mL glucose solution was prepared, and 18 10mL test tubes were prepared and divided into two groups, two of which were numbered 1, 2, 3, 4, 5, 6, and 7. Measuring the reagents according to the table 1, adding 2mL of 3, 5-dinitrosalicylic acid (DNS) reagent into each test tube, boiling for 2min in boiling water, cooling by running water, fixing the volume to 10mL by using ultrapure water, shaking up, and measuring the OD value of each test tube at 490nm wavelength by taking the test tube No.1 as a reference. With A_490nmThe lower absorbance is the abscissa and the content of glucose (mg/mL) is the ordinate, and a glucose standard curve is plotted.

TABLE 1 glucose Meter calibration Curve

1.3.3 determination of cellulase Activity

And (3) respectively detecting the activities of the cellulose endoglucanase (CMCase), the filter paper enzyme (FPA) and the cellulose exoglucanase (CX) by using the inactivated crude enzyme solution as a control through a DNS method. The enzyme activity of the cellulose endoglucanase is measured by adding 0.2mol/L of 1% CMC-Na in a 15mL test tube, taking sterile sodium acetate buffer solution with pH 4.8 as a substrate, adding 1mL of corresponding crude enzyme solution and 2mL of sodium acetate buffer solution, carrying out water bath at 50 ℃ for 30min, quantifying to 10mL with ultrapure water after completion, shaking up, taking the inactivated crude enzyme solution as a control, and measuring the generation amount of reducing sugar by adopting a DNS method.

In the determination of the filter paper enzyme and the cellulose exonuclease enzyme activity, 0.2mol/L of a filter paper strip of 1cm × 3cm, a sterile sodium acetate buffer solution with the pH value of 4.8 and 0.2mol/L of 0.05g of absorbent cotton and a sterile sodium acetate buffer solution with the pH value of 4.8 are respectively used as substrates, and the determination is carried out according to the method^[7]。

Definition of enzyme activity: under the conditions of certain temperature and pH, the enzyme amount required for enabling a substrate to generate 1 mu mol of glucose in 1min is one enzyme activity unit and is expressed by IU/mL.

The enzyme activity calculation formula is as follows: enzyme activity (IU) is the amount of reducing sugar × dilution times × 1000 ×/180 × 0.5 × reaction time

1000-enzyme activity unit conversion factor

180-glucose molecular weight

1.3.4 determination of the growth Curve of the Strain

Inoculating CX10 strain in 100 mM MC-Na culture medium at 5% inoculation amount, culturing at 37 deg.C after 4 passages, and measuring bacterial liquid OD every 6h_600nmThe growth curve of the strain was plotted by running 3 groups in parallel for 48h in a row. Measuring the inoculum size curve, setting the inoculum size (v/v) to be 2%, 4%, 6%, 8%, 10% and 12%, and measuring the OD of the bacterial liquid after 24h growth_600nmEach group was paralleled 3 times and an inoculum curve was plotted.

1.4 identification of the type of cellulase-producing Strain

1.4.1 morphological characteristics of the Strain

Smearing the separated and purified strains by adopting gram staining, capsule staining and spore staining methods respectively, and observing the microscopic staining forms of the strains.

1.4.2 Biochemical identification experiments

Biochemical identification is carried out on the separated and purified strains by adopting a VITEK2 microbial biochemical automatic identification instrument and a enterobacteriaceae biochemical identification tube, wherein the enterobacteriaceae biochemical identification tube method refers to Dongxu pearl and other 'common bacteria system identification manuals'.

1.4.316SrRNA gene identification

Extracting the genome DNA of the fiber degrading bacteria, and amplifying a 16SrDNA complete sequence gene by using bacterial universal 16SrDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3'). The total reaction volume was 25. mu.L, template DNA 2.0. mu.L, PCRPermix 12.5. mu.L, Forwardperfect 1.0. mu.L, Reverreprimer 11.0. mu.L, 16s-freeH₂O8.5. mu.L. The PCR amplification conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, renaturation at 72 ℃ for 1.5min and 30 cycles; finally, extension was carried out at 72 ℃ for 5 min. The amplification product was detected by 1% agarose gel electrophoresis, and pictures were taken under an ultraviolet lamp after EB staining. And storing the amplification product at-20 ℃, and sending the amplification product to Shanghai Producer gene sequencing company for detection.

2 results and analysis

2.1 screening of cellulase-producing strains

As shown in Table 2, 4 kinds of media including CMC-Na medium, LB medium, TSB and blood plate were used to perform strain primary screening of termite enterobacteria under aerobic condition at 37 ℃, 10 kinds of bacteria with good growth state were selected, the ratio (D/D) of the transparent hydrolysis ring of the strain to the diameter of the colony was measured, and the bacteria isolated from termite intestinal tract were named CX1-CX10 in sequence. Referring to FIG. 1, after Congo red staining, it can be observed that the bacteria have obvious hydrolysis ring on CMC-Na culture medium, and in addition, 2 bacteria are obtained by co-separation on 4 culture media under anaerobic environment, but no water hydrolysis ring is observed obviously, so no mark is made.

TABLE 2 morphological characteristics of Termite gut bacterial colonies

2.2 determination of cellulase Activity

As shown in fig. 2, a glucose standard curve is prepared according to the DNS method, and the linear equation of the obtained standard curve is y-5.1196 x +0.0441, and the correlation coefficient R is²0.9916, the linear correlation was good, and it was used as a cellulase activity assay for the isolated termite enteric bacteria.

2.3 determination of enzyme Activity

Respectively inoculating 10 strains separated and screened from termite intestinal tracts into a CMC-Na liquid fermentation medium, performing shake culture at 37 ℃ and 150rpm, respectively measuring cellulose endonuclease (CMCase), filter paper enzyme activity (FPA) and cotton enzyme activity (CX) in 48h, and further screening according to the enzyme activity. As can be seen from fig. 3: among 10 strains, the CMCase strains with higher enzyme activity are CX8, CX9 and CX10 strains, and the activities are 67.8IU/mL, 70.4IU/mL and 95.0IU/mL respectively; the FPA has the highest enzyme activity of CX10 strain, and the activity is 49.51 IU/mL; the CX enzyme activity is CX8, CX9 and CX10 which are higher, and the activity is 20.96IU/mL, 25.23IU/mL and 23.26IU/mL respectively. By synthesizing the superposition result of the activities of 3 cellulase, CX10, CX8 and CX9 are higher than other strains, which shows that the synergistic effect in the cellulase system is stronger, and the acting capability on multi-component cellulose substrates is high, wherein the activity of CX10 filter paper is obviously higher than that of CX8 and CX9, which shows that the decomposition capability on the cellulose substrates containing more crystallization regions is stronger than that of other strains. The analysis shows that 3 kinds of enzyme activities are different due to different substrates of the same strain, and the enzyme activities of different strains are obviously different. Since the 3 enzyme activities of the strain CX10 were the highest, strain CX10 was selected as a subject of subsequent studies.

2.3 identification of cellulase-producing strains

2.3.1 morphological characteristics

Single colonies CX2, CX3, CX4, CX5, CX8, CX9 and CX10 smears separated and purified from termite intestines are fixed on an alcohol lamp and are respectively subjected to gram staining, wherein CX2, CX3, CX4 and CX5 are gram-negative bacilli, and CX8, CX9 and CX10 are gram-positive bacilli. Meanwhile, CX10 was further observed by using the methods of capsular staining and spore staining, and the results are shown in FIG. 6, the strain is gram-positive bacillus, and has no capsule, and endophytic spores are visible in spore staining.

2.3.2 Biochemical Experimental identification

According to the identification result of the morphological characteristics of the bacteria 2.3.1, the strains CX2, CX3, CX4 and CX5 were biochemically identified by using a biochemical identification tube of Enterobacteriaceae. As shown in Table 3, the biochemical properties of CX2, CX3, CX4 and CX5 were not completely consistent between the indigo substrate and the citrate detection. Referring to the handbook of common bacteria systems, CX2 and CX4 were preliminarily judged to be Serratia (Serratia) of Enterobacteriaceae, and CX3 and CX5 are Citrobacter (citrobacter) of Enterobacteriaceae. Meanwhile, the automatic biochemical determinator for the microorganisms of VITEK2 is adopted to analyze and identify the biochemical characteristics of CX8, CX9 and CX 10. As shown in Table 4, the biochemical properties of CX8, CX9 and CX10 were different among Amygdalin (AMY), D-sorbitol (dSOR), β -D-glucuronidase (BGUR) and D-mannitol (dMAN). According to Bergey's Manual of systematic bacteriology, CX8, CX9 and CX10 are preliminarily judged to be Bacillus (Bacillus) belonging to Bacillaceae.

TABLE 3 Biochemical identification of the Enterobacteriaceae CX2, CX3, CX4 and CX5 bacteria

TABLE 4 Biochemical identification results of Bacillus bacteria CX8, CX9 and CX10

2.3.316 PCR amplification of SrDNA and construction of phylogenetic tree

The DNA of CX2, CX3, CX4, CX5, CX8, CX9 and CX10 strains are respectively extracted, the 16SrDNA gene is amplified by adopting the bacterial universal primers 27F and 1492R, the size of the target fragment is about 1500bp, and the amplification result is shown in FIG. 5.

Sequencing the 16SrDNA gene amplification products of each strain obtained by cloning, and carrying out BLAST comparison in an NCBI database. As a result, as shown in FIG. 6, when the strains having high homology with the obtained sequences were compared in GenBank, it was found that CX2 and CX4 both originated from the gene sequence of Serratia (Serratia) and the identity was more than 95%. CX3 and CX5 are both derived from the gene sequence of Citrobacter (citrobacter) and have the consistency of more than 95 percent, so that the strain is judged to belong to the Citrobacter (citrobacter). The strains CX8, CX9 and CX10 are all derived from the gene sequence of Bacillus (Bacillus) and have homology higher than 95%, wherein the strains CX8 and CX10 have homology with Bacillus firmus (Bacillus firms) as high as 97%, and the strain CX9 has homology with Bacillus sp.171544 as high as 96%.

In the research, low-grade wood yellow mealworm termites are taken as a research object, and the intestinal strains of the termites separated and purified by adopting an aerobic culture method mainly comprise bacillus, citrobacter and serratia, which are similar to the research results of discovering the intestinal microbial flora of the termites by Haizhuzhoo and the like. Meanwhile, Xurong et al adopt Congo red solid plate method to separate and purify bacillus amyloliquefaciens and bacillus subtilis from higher wood termite intestinal microorganisms. Thayer isolated Bacillus cereus (Bacillus cereus) from the hindgut of Reticulitermes spp (Reticulitermes fisherus) which produces carboxymethylcellulase activity. However, the test uses CMC-Na as separation medium and CMC-Na congo red as identification medium, and separates 10 strains from the intestinal tract of Braytonema arborescens through aerobic and anaerobic culture methods, and the dominant strain CX8 separated under aerobic condition is separated for separating the strains under anaerobic condition, and is identified as strong bacillus through 16SrRNA gene, and is not reported in the prior literature. In this study, no genera such as Cellophilus, enterococcus, and Clostridium thermocellum which appear in the termite gut were isolated, which may be related to the facultative organisms in the termite gut to scavenge oxygen and create anaerobic conditions, or may be related to the culture medium and culture conditions selected in the experiment. In addition, the species of termites can also be an important factor. According to whether the hindgut of the termites contains symbiotic protists or not, the termites can be divided into two major groups, namely low-class termites (with symbiotic protists) and high-class termites (without symbiotic protists), the termites purchased in the research are woodmeal yellow thorny termites, belong to the low-class termites, the species of bacteria in the intestinal tracts are very rich, but most of the bacteria are non-culturable microorganisms, and the hindgut bacteria and archaea mainly exist in cytoplasm and the outer surface of flagellates. The diversity study of lower termite intestinal bacteria by methods such as 16S rRNA sequencing and the like shows that the main groups of the lower termite intestinal symbiotic bacteria are spirochaetes (Spirochetes), Bacteroidetes (bacteroides), Firmicutes (Firmicutes), traceable bacteria (Elusiicrobia) and Proteobacteria (Proteobacteria). The PieerHethner separates an anaerobic clostridium clostridia mitondissp.nov.from higher termites and has the capacity of degrading cellulose. Therefore, the 16SrRNA gene library sequence, the genomics and the like of the intestinal microorganisms are analyzed by a non-culture method, and the problems that a plurality of microorganisms cannot be cultured at present and strains degenerate, mutate, pollute and the like in the culture and purification process can be solved.

According to the research, carboxymethyl cellulose is used as a matrix, an enrichment culture medium is established, and after Congo red staining solution is used, strains CX1-CX10 with high cellulase yield are preliminarily screened by comparing the ratio of the transparent hydrolysis circle of each bacterium to the diameter of a bacterial colony. The cellulase can be divided into three types of exo-beta-1, 4-glucosidase, endo-beta-1, 4-glucosidase and beta-1, 4-glucolase, and the test further screens bacteria separated and purified from the intestinal tracts of termites by measuring the enzyme activities of three enzyme systems of cellulose endo-cellulase (CMCase), filter paper enzyme activity (FPA) and cotton enzyme activity (CX) in the strains and superposing the three enzyme activities for comparison. As a result, the 3 enzyme activities of CX8 strain within 48h are found to be advantageous. The screened termite intestinal cellulose degradation strain CX8 has high biodiversity value in research, and has necessary conditions for preparing strains for converting lignocellulose into bioethanol and applying traditional Chinese medicine fermentation biotechnology. In addition, hydrolytic enzymes are also essential to the realization of conversion of lignocellulose into biofuels or application to the traditional Chinese medicine waste recycling technology. Therefore, the research needs to analyze and compare the biochemical and physiological properties of the hydrolytic enzymes in the strains to further determine the strains with good lignocellulose-degrading function in the termite intestinal microorganisms.

Bacillus in the termite gut plays an important role in the breakdown of lignocellulose. The bacillus CX8 is discovered from intestinal tracts of lower-grade woodmeal yellow thorny termites, the strain can produce cellulose endonuclease (CMCase), filter paper enzyme activity (FPA) and cotton enzyme activity (CX) within 48 hours, and the strong bacillus-CX 10 with the highest enzyme activity has further deep research value.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. A bacillus firmus CX8 from termite intestinal tract is characterized in that the strain is preserved in Beijing 3 Chaoyang district Beichen No.1 Hospital No. 3 China microorganism culture Collection common microorganism center in 8 months and 27 days in 2018, and the preservation numbers are as follows: CGMCC No. 16342.

2. The Bacillus firmus CX8 from the intestinal tract of termites, wherein the strain is gram positive bacilli, has no capsule, spores are visible when stained with endospores, colonies are round and single, colonies are white in color, and the edges of the colonies are intact.

3. The Bacillus firmus CX8 from the intestinal tract of termites, according to claim 1, wherein the strain is a cellulase-producing facultative anaerobic strain; the CMCase enzyme activity of the strain is 67.8IU/mL within 48h, and the CX enzyme activity is 20.96 IU/mL.

4. The development and application of the bacillus firmus CX8 in the cellulose-enriched crops and the traditional Chinese medicine fermentation recycling technology according to claim 1.

5. The use of the Bacillus firmus CX8 of claim 1 in the conversion of lignocellulose to bioethanol.

6. The use of the bacillus firmus CX8 according to claim 1 in the fermentation and reuse of Chinese herbal medicine waste.

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