CN115418337B - Lignin degrading bacterium and application thereof in rice straw micro-storage - Google Patents
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Abstract
A lignin degrading bacterium and application thereof in rice straw micro-storage relate to the field of rice straw forage treatment and aim to solve the technical problems of high energy consumption and secondary pollution of the existing method for reducing lignin in straw. The lignin degrading bacterium is Siamese bacillus (Bacillus siamensis) STQ-LIG, and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M20221169. The lignin degrading bacteria can be utilized to carry out rice straw micro-storage so as to degrade lignin and cellulose of the rice straw. The lignin degrading bacterial liquid is utilized to ferment rice straw, acid washing lignin, neutral washing fiber and acid washing fiber in a fermentation product are reduced, the content of volatile fatty acid is improved, the digestibility of in vitro dry matters is improved, and the method can be used in the field of straw micro-storage.
Description
Technical Field
The invention relates to the field of rice straw forage treatment, in particular to lignin degrading bacteria screened from horse manure and straw micro-storage application thereof.
Background
The annual crop straw of China is 7-8 hundred million tons. The annual yield of rice straw is more than 2 hundred million tons, and the crop straw is used as rich feed resources in China, so that the method has great potential for the utilization of feed for ruminant livestock. However, the widespread biochemical characteristics of straw have limited the applications in production. Firstly, the digestibility is low, and generally only 35% -50%. Secondly, the crude protein content is low, the crude protein content is 4% -5%, most of the crude protein is connected with the cell wall, the digestibility and the degradation rate are low, and sometimes the digestible crude protein is negative. Thirdly, the content of mineral elements is greatly changed by agriculture and soil pollution, wherein the content of silicon is higher, and the content of the mineral elements is inversely related to the degradation of polysaccharide in the straw. Fourthly, the cellulose and lignin content is high, lignin is a macromolecular polymer composed of polyphenol compounds, is an amorphous three-dimensional macromolecular compound with complex, stable and various structures, is combined with hemicellulose in a covalent bond form, embeds cellulose molecules therein to form a natural barrier, so that microorganisms are not easy to contact with the cellulose molecules, and the utilization rate and the nutritive value of straw are reduced. The traditional nutrition considers that the lignin content is inversely proportional to the digestibility of the straw, and the digestibility of animals can be improved by 4% -5% when the daily ration is reduced by 1%, so that lignin degradation is the key for improving the straw utilization efficiency in the straw treatment process, and common methods for breaking lignin and cellulose include radiation, steam explosion, puffing, grinding, acidolysis, alkali treatment, oxidation treatment, organic solvent method and the like, and although only about 50% of lignin in plant fiber raw materials can be removed by the traditional physical and chemical methods, a series of problems such as high energy consumption, secondary pollution, cost, livestock feed quality safety and the like are brought. Therefore, the search for safe biological degradation of lignin is an important way for straw feed utilization, not only can reduce environmental pollution and save energy consumption, but also can change waste into valuables and realize straw resource recycling.
Disclosure of Invention
The invention aims to solve the technical problems of high energy consumption and secondary pollution of the existing method for reducing lignin in straw, and provides a lignin degrading bacterium which is used for rice straw micro-storage, can solve the problem that lignin and cellulose are difficult to degrade in the micro-storage process of the dry rice straw, and improves the micro-storage fermentation quality of the straw.
The lignin degrading bacterium is Siamese bacillus (Bacillus siamensis) STQ-LIG, and is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation address is university of Wuhan, the preservation date is 2022, 7 months and 25 days, and the preservation number is CCTCC NO: M20221169.
The application of the lignin degrading bacteria is that the Siamese bacillus (Bacillus siamensis) STQ-LIG is utilized to carry out rice straw micro-storage to degrade lignin and cellulose of the rice straw.
Furthermore, the method for carrying out rice straw micro-storage by utilizing lignin degrading bacteria comprises the following steps:
adding lignin degrading bacterial liquid into rice straw, fermenting at 20-30 deg.c to complete micro storage of rice straw.
Further, the concentration of the Siamese bacillus (Bacillus siamensis) STQ-LIG in the lignin degrading bacterial solution is 1 multiplied by 10 9 ～5×10 9 cfu/mL, the additive amount of the lignin degrading bacterial liquid is 1% -5% of the dry weight of the rice straw.
Further, the fermentation is liquid fermentation or solid fermentation, and the fermentation condition is anaerobic fermentation.
Further, under the condition of solid state fermentation of rice straw, the water content of the rice straw is controlled to be 60-70%.
Further, the solid state fermentation time of the rice straw is 5-6 weeks.
The invention has the beneficial effects that:
the lignin degrading bacterium, namely Siamese bacillus (Bacillus siamensis) STQ-LIG, has the functions of degrading acidic washing lignin, neutral washing fiber and acidic washing fiber in the processes of liquid fermentation and solid micro-storage of rice straw, improves the digestion rate of dry matters in vitro and the content of short-chain volatile fatty acids in the rice straw, and reduces the crystallinity of the fiber.
After the lignin degrading bacteria are utilized to sterilize the rice straw by liquid fermentation treatment for 2 weeks, compared with a control, the contents of acid washing lignin, neutral washing fiber and acid washing fiber of the rice straw in the bacterial liquid treatment group are respectively reduced by 17.93%, 10.46% and 5.45%; the degradation rate of the acidic washing lignin and neutral washing fiber is 4.65 times and 2.42 times of that of the control group respectively; the lignin degrading bacteria show strong capacity of degrading acidic lignin washing and neutral fiber washing of rice straw.
After the strain is used for carrying out solid fermentation treatment on the non-sterilized rice straw for 6 weeks, compared with a control group, the contents of acidic washing lignin, neutral washing fiber and acidic washing fiber of the rice straw are respectively reduced by 20.93%, 1.82% and 3.72%; the degradation rate of the acidic washing lignin and neutral washing fiber is 2.35 times and 9.68 times that of the control group respectively; the digestibility of dry matters in vitro of rice straws for 12, 24, 48 and 72 hours is improved by 12.28 percent, 10.82 percent, 19.00 percent and 13.84 percent; the fermentation product has increased volatile fatty acid content such as acetic acid, propionic acid and butyric acid compared with control.
The Siamese Bacillus (Bacillus siamensis) STQ-LIG is Siamese Bacillus, belongs to Bacillus and is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is university of Wuhan, the preservation date is 2022, 7 months and 25 days, and the preservation number is CCTCC NO: M20221169.
Drawings
FIG. 1 is a phylogenetic tree of the Siamese bacillus (Bacillus siamensis) STQ-LIG constructed in accordance with the present invention;
FIG. 2 is an X-ray diffraction (XRD) spectrum of rice straw before and after micro-storage in example 3.
Detailed Description
The following examples are used to demonstrate the benefits of the present invention.
Example 1: the lignin degrading bacterium of the embodiment, namely the Siamese bacillus (Bacillus siamensis) STQ-LIG screening and obtaining method, comprises the following steps:
10g of freshly discharged horse dung is put into a 250mL conical flask, 90mL of sterile water is added, and the mixture is subjected to constant temperature oscillation for 30min at the temperature of 30 ℃ at the rotating speed of 120r/min to obtain suspension; 1mL of the suspension was aspirated with the sterilized tip for 10 -1 -10 -9 Serial cell concentration gradient dilutions were performed, 10 -1 ，10 -3 ，10 -5 ，10 -7 ，10 -9 Respectively taking 0.3mL of 5 gradient dilutions on a separation culture medium, and placing the separation culture medium in a constant temperature incubator at 28 ℃ for inversion for 3 days; after the colony is grown out of the flat plate, according to the morphological characteristics of the colony, a single colony is picked by an inoculating loop and inoculated in a solid LB culture medium, and the single colony is repeatedly streaked out. Streaking the strain screened in solid LB culture medium at 28deg.C for 24 hr, selecting small amount of strain, inoculating in 50mL sterilized liquid LB culture medium conical flask for strain rejuvenation, and transferring with transfer gunTaking 5 mu L of rejuvenating bacterial liquid, uniformly inoculating the bacterial liquid into an aniline blue culture medium and a sodium carboxymethyl cellulose culture medium, culturing for 72 hours at 28 ℃ in an inverted mode, checking the sizes of colony hydrolysis circles on the aniline blue culture medium and the sodium carboxymethyl cellulose culture medium, and selecting out bacterial colonies with larger colony hydrolysis circles of the aniline blue culture medium and the sodium carboxymethyl cellulose culture medium from the bacterial colonies, namely the bacterial strains with lignin and cellulose degradation capacity, wherein 16s rDNA is identified as bacillus siamensis (Bacillus siamensis), the bacterial strains are named bacillus siamensis (Bacillus siamensis) STQ-LIG, the acquisition time is 2022 and the acquisition time is 1 month and 25 days, and the sites are: northeast geography and agricultural ecological institute of China academy of sciences in Harbin, heilongjiang province.
Wherein the medium is isolated: (NH 4) 2 SO 4 2g，MgSO 4 ·7H 2 O 0.5g，K 2 HPO 4 1g, 0.5g of NaCl, 5g of alkaline lignin, 22g of agar powder, 1L of distilled water for constant volume, 121 ℃ and sterilization for 30min.
LB medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 20.0g of agar, 1L of distilled water for constant volume, adjusting the pH to 7.0-7.4, sterilizing at 121 ℃ for 30min.
Aniline blue medium: 0.1g of aniline blue is dissolved in 10mL of distilled water to obtain aniline blue dye liquor; then 10.0g yeast extract powder, 20.0g glucose, 15.0g agar and 990mL distilled water are taken to prepare a culture medium, the culture medium is sterilized, a disposable medical injector is used for sucking aniline blue dye liquid, a sterile filter is arranged and injected into the sterilized culture medium, and the culture medium is mixed uniformly in an ultra clean bench when the culture medium is hot.
Sodium carboxymethyl cellulose medium: CMC-Na20g, NH 4 NO 3 1.0g，KH 2 PO 4 1.5g，MgSO 4 ·7H 2 O0.5g，Na 2 HPO 4 2.5g, yeast extract 0.5g, peptone 2.5g, agar 15.0g, distilled water to a volume of 1L, pH 7.0-7.2, and autoclaving at 121 ℃ for 30min.
The Siamese bacillus (Bacillus siamensis) STQ-LIG screened in the embodiment is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is university of Wuhan, the preservation date is 2022, 7 months and 25 days, and the preservation number is CCTCC NO: M20221169.
The Siamese bacillus (Bacillus siamensis) STQ-LIG of this example was grown in solid LB medium.
The morphology and molecular characterization of the screened Siamese bacillus (Bacillus siamensis) STQ-LIG were as follows.
(1) Morphological characteristics of Siamese bacillus STQ-LIG:
the Siamese bacillus (Bacillus siamensis) STQ-LIG screened in the example shows milky white, opaque, surface wrinkles, irregular edges, tilted edges, inward middle and sticky surface on the solid LB culture medium. Gram positive bacteria are in the shape of a rod.
(2) Molecular identification:
1) Genomic DNA extraction
The Siamese bacillus (Bacillus siamensis) STQ-LIG strain is inoculated in a liquid LB culture medium and is subjected to shaking culture at 28 ℃ for 48 hours to obtain a bacterial suspension. The bacterial suspension was centrifuged, the supernatant was discarded, washed with sterile water and centrifuged again, the supernatant was discarded to obtain pure bacterial cells, and then genomic DNA was extracted according to the genomic DNA purification kit (Promega) instructions.
2) 16s rDNA PCR amplification
The target gene fragment was amplified using bacterial universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'). The PCR reaction system is shown in Table 1
TABLE 1 PCR reaction System
| Reaction system | μL |
| PCR Mix | 21 |
| Primer F(5p) | 1 |
| Primer R(5p) | 1 |
| Stencil (ng/ul) | 2 |
| Totalizing | 25 |
The reaction conditions are as follows: pre-denaturation at 96 ℃ for 5min;96 ℃ 20sec,62 ℃ 30sec,72 ℃ 30sec, and the cycle number is 35; finally, the mixture is extended for 10min at 72 ℃ and stored at 4 ℃.
3) PCR product detection and purification
The 3. Mu.L PCR products were subjected to 1.0% agarose gel electrophoresis, and the band properties were observed.
The PCR product purification is operated according to the operation flow of the magnetic bead purification standard in GB/T40171-2021 general rules for detection of magnetic bead method DNA extraction and purification kit.
The resulting sequenced sequence was subjected to BLAST (National Center for Biotechnology Information [ http:// www.ncbi.nlm/nih.gov ]) homology alignment with the nucleic acid data in GenBank, and the results showed that the strain STQ-LIG had 99.86% homology with Bacillus siamensis KCTC 13613, and a phylogenetic tree was constructed using software MOLECULAR EVOLUTIONARY GENETIC ANALYSIS software (MEGA 5.0), as shown in FIG. 1, and the Bacillus siamensis KCTC 13613 sequence constituted a stable evolutionary branch, and thus was designated Bacillus siamensis STQ-LIG.
Example 2: the Siamese bacillus (Bacillus siamensis) STQ-LIG screened in the example 1 is utilized to carry out micro-storage on sterilized rice straws, and the degradation effect on lignin and cellulose is examined. The method comprises the following steps:
1. culturing of the Siamese bacillus (Bacillus siamensis) STQ-LIG Strain:
liquid LB medium: 5g of yeast extract powder, 10g of peptone and 10g of NaCl, adding water to 1000mL, pH7.0-7.4, and sterilizing at 121 ℃ for 20min.
Selecting a single colony of purified Siamese bacillus (Bacillus siamensis) STQ-LIG, inoculating the single colony into a liquid LB culture medium, and culturing the single colony for 48 hours on a shaking table at a constant temperature of 28 ℃ at 200rpm/min to obtain Siamese bacillus (Bacillus siamensis) STQ-LIG bacterial liquid for later use; the concentration of bacteria in the bacterial liquid is about 1 multiplied by 10 9 cfu/ml。
2. Weighing 0.6g of sterilized rice straw powder, sterilizing in a centrifuge tube at 121 ℃, and then adding 40mL of sterilized inorganic salt culture medium to form a liquid fermentation culture medium; wherein the inorganic salt culture medium is as follows: KH (KH) 2 PO 4 1.0g,NaCl 0.1g,CaCl 2 0.1g,MgSO 4 ·7H 2 O 0.3g,NaNO 3 2.5g,FeCl 3 0.1g, distilled water 1L constant volume, pH to 7.0-7.4,121 ℃ and sterilizing for 30min. 0.4mL of Siamese bacillus (Bacillus siamensis) STQ-LIG bacterial liquid (1% of inoculation amount) is inoculated into a centrifugal tube respectively, the inoculated centrifugal tube is placed into a shaking table in an inclined way, and micro-storage is carried out under the conditions of rotating speed of 180rmp and temperature of 28 ℃; 3 replicates were set for each strain, and a control group without siamese bacillus (Bacillus siamensis) STQ-LIG was set up. After 2 weeks of culture, taking out the sample, centrifuging, discarding the supernatant, filtering to obtain a fermented sample, weighing the fermented weight, and calculating the loss rate of the straw. And (3) drying the fermented sample, measuring the content of the fermented sample NDF, ADF and ADL, and calculating the degradation rate of the fermented sample NDF, ADF and ADL.
The results show that: after the Siamese bacillus (Bacillus siamensis) STQ-LIG bacteria treat rice straws for 2 weeks through liquid fermentation, compared with a blank control group, the Siamese bacillus (Bacillus siamensis) STQ-LIG bacteria liquid treat rice straws, neutral washing fiber (NDF), acid washing fiber (ADF) and acid washing lignin (ADL) contents are reduced, and meanwhile, the degradation rates of the NDF, the ADF and the ADL are obviously increased, the control group is respectively 7.82%, 6.21% and 4.35%, and the bacteria liquid treatment group is respectively 18.93%, 6.38% and 20.22%, as shown in Table 2.
Table 2 Siamese bacillus (Bacillus siamensis) STQ-LIG bacterium effect on degradation of lignin and cellulose in sterilized Rice straw (Dry basis)
Example 3: the siamese bacillus (Bacillus siamensis) STQ-LIG screened in the example 1 is utilized to carry out micro-storage on the unsterilized rice straw, and the degradation effect on lignin and cellulose is examined. The method comprises the following steps:
1. culturing of the Siamese bacillus (Bacillus siamensis) STQ-LIG Strain:
liquid LB medium: 5g of yeast extract powder, 10g of peptone and 10g of NaCl, adding water to 1000mL, pH7.0-7.4, and sterilizing at 121 ℃ for 20min.
Selecting a single colony of purified Siamese bacillus (Bacillus siamensis) STQ-LIG, inoculating the single colony into a liquid LB culture medium, and culturing the single colony for 48 hours on a shaking table at a constant temperature of 28 ℃ at 200rpm/min to obtain Siamese bacillus (Bacillus siamensis) STQ-LIG bacterial liquid for later use; the concentration of bacteria in the bacterial liquid is about 1 multiplied by 10 9 cfu/ml。
2. Weighing 50g of rice straw in a fermentation bag, adding 2.5mL of Siamese bacillus (Bacillus siamensis) STQ-LIG bacterial liquid and 90mL of distilled water, keeping the moisture content of the rice straw within 62% -66%, vacuumizing, sealing the fermentation bag, repeating each treatment for three times, weighing the sample after 6 weeks, and measuring the influence of bacterial liquid micro-storage on chemical components of the rice straw.
The results show that: after the Siamese bacillus (Bacillus siamensis) STQ-LIG bacterial liquid is fermented and treated on the non-sterilized rice straw for 6 weeks, compared with a blank control group, the contents of acidic washing lignin, neutral washing fiber and acidic washing fiber of the rice straw are respectively reduced by 20.93%, 1.82% and 3.72%; the degradation rates of the acid washing lignin and the neutral washing fiber are 2.35 times and 9.68 times that of the control group respectively, as shown in table 3; the fermentation product had increased amounts of volatile fatty acids such as acetic acid, propionic acid and butyric acid compared to the control, as shown in table 4; the digestibility of dry matter in vitro of rice straw 12, 24, 48 and 72 hours was improved by 12.28%, 10.82%, 19.00% and 13.84%, as shown in table 5, and the fiber crystallinity was reduced as seen in fig. 2 by X-ray diffraction analysis of the XRD spectrum of the fiber, as shown in fig. 2.
Table 3 Siamese bacillus (Bacillus siamensis) STQ-LIG bacterium degradation effect (dry matter basis) on lignin and cellulose of unsterilized rice straw
| Detecting items | Blank control group | Bacterial liquid treatment group |
| NDF mass percent (%) | 75.46 | 74.09 |
| ADF mass percent (%) | 51.68 | 49.76 |
| ADL mass percent (%) | 10.94 | 8.65 |
| NDF degradation rate (%) | 0.44 | 4.26 |
| ADF degradation Rate (%) | -0.45 | 0.65 |
| ADL degradation rate (%) | 6.61 | 15.56 |
TABLE 4 influence of Siamese bacillus (Bacillus siamensis) STQ-LIG on Rice straw fermentation products
| Detecting items | Blank control group | Bacterial liquid treatment group |
| pH | 4.59 | 4.66 |
| Ammonia nitrogen g/kg | 1.16 | 1.10 |
| Lactic acid g/kg | Not detected | Not detected |
| Acetic acid g/kg | 28.61 | 33.51 |
| Propionic acid g/kg | 2.26 | 4.53 |
| Butyric acid g/kg | 30.44 | 43.53 |
TABLE 5 influence of Siamese bacillus (Bacillus siamensis) STQ-LIG on in vitro digestibility of rice straw
| Detecting items | Blank control group | Bacterial liquid treatment group |
| 12h in vitro digestibility (%) | 21.50 | 24.14 |
| 24h in vitro digestibility (%) | 35.66 | 39.52 |
| 48h in vitro digestibility (%) | 40.89 | 48.66 |
| 72h in vitro digestibility (%) | 45.73 | 52.06 |
Claims (8)
1. A lignin degrading bacterium is characterized in that the lignin degrading bacterium is Siamese bacillusBacillus siamensis) STQ-LIG is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is university of Wuhan, the preservation date is 2022, 7 months and 25 days, and the preservation number is CCTCC NO: M20221169.
2. The use of a strain of lignin-degrading bacteria according to claim 1 wherein the use is made of Bacillus siamensis @Bacillus siamensis) STQ-LIG is used for rice straw micro-storage.
3. The application of the lignin degrading strain according to claim 2 wherein the method for rice straw micro-storage by using lignin degrading strain comprises the following steps:
adding lignin degrading bacterial liquid into rice straw, fermenting at 20-30 deg.c to complete micro storage of rice straw.
4. The application of the lignin-degrading bacterium according to claim 3, wherein the lignin-degrading bacterium is characterized in that the Bacillus siamensis is @ in the lignin-degrading bacterium solutionBacillus siamensis) STQ-LIG concentration of 1×10 9 ～5×10 9 cfu/mL, the additive amount of the lignin degrading bacterial liquid is 1% -5% of the dry weight of the rice straw.
5. The use of a strain of lignin degrading bacteria according to claim 3 or 4 wherein the rice straw is sterilized rice straw or non-sterilized rice straw.
6. The use of a strain of lignin-degrading bacteria according to claim 3 or 4 wherein the fermentation is either liquid fermentation or solid fermentation, the fermentation conditions being anaerobic.
7. The application of the lignin degrading strain according to claim 3 or 4 wherein the moisture content of rice straw is controlled between 60% and 70% by mass under the condition of solid state fermentation of rice straw.
8. The use of a lignin degrading strain according to claim 3 or 4 wherein the time for solid state fermentation of rice straw is between 5 and 6 weeks.
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