CN115418337A - Lignin degrading bacterium and application thereof in rice straw micro-storage - Google Patents
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Abstract
A lignin degrading bacterium and an application thereof in rice straw micro-storage relate to the field of rice straw feed processing and aim to solve the technical problems of high energy consumption and secondary pollution of the existing method for reducing lignin in straws. The lignin degrading bacteria of the invention are Siamese Bacillus (Bacillus siemensis) STQ-LIG which are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M20221169. The lignin degrading bacteria can be used for micro-storage of the rice straws so as to degrade lignin and cellulose of the rice straws. The lignin-degrading bacteria liquid is utilized to ferment the rice straws, so that the acidic washing lignin, the neutral washing fiber and the acidic washing fiber in the fermentation product are reduced, the content of volatile fatty acid is improved, the digestion rate of dry matters in vitro 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 a lignin-degrading bacterium screened from horse manure and application of the lignin-degrading bacterium to straw micro-storage.
Background
The annual crop straw yield of China is 7-8 hundred million tons. Wherein the annual output of the rice straws is more than 2 hundred million tons, and the crop straws are used as rich feed resources in China and have great potential for the utilization of ruminant livestock feed. However, the general biochemical characteristics of straw have limited application in production. Firstly, the digestibility is low, and is generally only 35 to 50 percent. Secondly, the content of crude protein is low, the content of crude protein is 4% -5%, most of the crude protein is connected with cell walls, the digestibility and the degradation rate are low, and the digestible crude protein is negative sometimes. And 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 silicon is negatively related to the degradation of polysaccharide in the straws. Fourthly, the content of cellulose and lignin is high, the lignin is a macromolecular polymer consisting of polyphenol compounds, is an amorphous three-dimensional macromolecule with a complex, stable and various structure, is combined with hemicellulose in a covalent bond mode, and embeds cellulose molecules in the macromolecular polymer to form a natural barrier, so that microorganisms are not easy to contact with the cellulose molecules, and the utilization rate and the nutritional value of the straw are reduced. The traditional nutriology holds that the lignin content is inversely proportional to the straw digestibility, and the animal digestibility can be improved by 4-5% when the lignin is reduced by 1% per day, so the lignin degradation is the key for improving the utilization efficiency of the straw in the straw treatment process, and common methods for breaking the lignin and the cellulose comprise radiation, steam explosion, puffing, grinding, acid hydrolysis, alkali treatment, oxidation treatment, organic solvent method and the like, and although the traditional physical and chemical methods can only remove about 50% of the lignin in plant fiber raw materials, the traditional physical and chemical methods bring a series of problems of high energy consumption, secondary pollution, cost, safe livestock feed quality and the like. Therefore, seeking an important way for degrading lignin by a safe biological method is an important way for straw feed utilization, not only can reduce environmental pollution and save energy consumption, but also can change waste into valuable 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 straws, and provides a strain of lignin degrading bacteria which is used for micro-storage of rice straws, can solve the problem that lignin and cellulose are difficult to degrade in the micro-storage process of withered and yellow rice straws, and improves the micro-storage fermentation quality of the straws.
The lignin degrading bacteria are Siamese Bacillus (Bacillus siemensis) STQ-LIG which are preserved in China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan university, the preservation date is 2022 years, 7 months and 25 days, and the preservation number is M20221169.
The application of the lignin degrading bacteria is that rice straws are subjected to micro-storage by utilizing Bacillus siamensis STQ-LIG so as to degrade lignin and cellulose of the rice straws.
Furthermore, the method for micro-storing the rice straws by using the lignin degrading bacteria comprises the following steps:
adding the lignin-degrading bacterial liquid into the rice straws, and fermenting at the temperature of 20-30 ℃ to finish the micro-storage of the rice straws.
Furthermore, the concentration of Siamese Bacillus (Bacillus siamensis) STQ-LIG in the lignin degrading bacterium liquid is 1 multiplied by 10 9 ～5×10 9 cfu/mL, and 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 state fermentation or solid state fermentation, and the fermentation condition is anaerobic fermentation.
Furthermore, under the condition of solid-state fermentation of the rice straws, the water content of the rice straws is controlled to be 60-70 percent by mass.
Furthermore, the time of the solid-state fermentation of the rice straws is 5-6 weeks.
The invention has the beneficial effects that:
the lignin degrading bacteria, namely Siamese Bacillus (Bacillus siamensis) STQ-LIG, have 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 straws, improve the in vitro dry matter digestibility and short-chain volatile fatty acid content of the rice straws and reduce the fiber crystallinity.
After the rice straws are sterilized by the lignin degrading bacteria through liquid fermentation for 2 weeks, compared with a control, the contents of acid washing lignin, neutral washing fiber and acid washing fiber of the rice straws of the bacteria liquid treatment group are respectively reduced by 17.93 percent, 10.46 percent and 5.45 percent; the degradation rates of the acidic washing lignin and the neutral washing fiber are respectively 4.65 times and 2.42 times of those of a control group; the lignin degrading bacteria show strong capability of degrading rice straw acid washing lignin and neutral washing fiber.
After the bacterial strain is subjected to solid fermentation treatment on unsterilized rice straws for 6 weeks, compared with a control group, the contents of acid washing lignin, neutral washing fiber and acid washing fiber of the rice straws are respectively reduced by 20.93 percent, 1.82 percent and 3.72 percent in bacterial liquid treatment; the degradation rates of the acidic washing lignin and the neutral washing fiber are respectively 2.35 times and 9.68 times of the degradation rates of the control group; the in vitro dry matter digestibility of the rice straws is improved by 12.28%, 10.82%, 19.00% and 13.84% in 12, 24, 48 and 72 hours; compared with the control, the fermentation product has increased content of volatile fatty acids such as acetic acid, propionic acid and butyric acid.
Siamese Bacillus (Bacillus siamensis) STQ-LIG belongs to Bacillus and is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan university, the preservation date is 2022 years, 7 months and 25 days, and the preservation number is CCTCC NO: M20221169.
Drawings
FIG. 1 is a phylogenetic tree of Siamese Bacillus (Bacillus siamensis) STQ-LIG constructed according to the present invention;
FIG. 2 is the X-ray diffraction (XRD) spectrum of rice straw in example 3 before and after micro-storage.
Detailed Description
The following examples are used to demonstrate the beneficial effects of the present invention.
Example 1: the method for screening and obtaining lignin degrading bacteria, namely Siamese Bacillus (Bacillus siamensis) STQ-LIG, comprises the following steps:
putting 10g of freshly discharged horse dung into a 250mL conical flask, adding 90mL of sterile water, and oscillating at the constant temperature of 30 ℃ for 30min at the rotating speed of 120r/min to obtain a suspension; sucking 1mL of suspension by using a sterilized gun head for 10 -1 -10 -9 Serial cell concentration gradient dilutions, 10 each -1 ，10 -3 ，10 -5 ，10 -7 ，10 -9 Concentration, 0.3mL of each of 5 gradient diluents is put on a separation culture medium and placed in a constant temperature incubator at 28 ℃ for inversion for 3 days; after the plate grows out of the colonies, selecting a single colony by using an inoculating loop according to the morphological characteristics of the colony, inoculating the single colony in a solid LB culture medium, and repeatedly streaking to grow a single colony. Marking the primarily screened strains into a solid LB culture medium, culturing for 24 hours at 28 ℃, then picking a small amount of strains, inoculating the strains into a 50mL conical flask of a sterilized liquid LB culture medium for strain rejuvenation, using a liquid transfer gun to transfer 5 mu L of the rejuvenated strains, uniformly dotting the strains in an aniline blue culture medium and a sodium carboxymethyl cellulose culture medium, performing inverted culture for 72 hours at 28 ℃, checking the sizes of colony hydrolysis rings on the aniline blue culture medium and the sodium carboxymethyl cellulose culture medium, selecting colonies with larger colony hydrolysis rings of the aniline blue culture medium and the sodium carboxymethyl cellulose culture medium, namely the strains are strains with lignin and cellulose degradation capacity, identifying the strains as Bacillus siamensis (Bacillus siamensis) through 16s rDNA, and the strains are named as Bacillus siamensis (Bacillus siamensis) STQ-LIG, wherein the obtaining time is 2022 years, 1 month and 25 days, and the sites are as follows: the institute of geography and agroecology in northeast of the academy of sciences in Harbin, heilongjiang province.
Wherein the separation culture medium: (NH 4) 2 SO 4 2g，MgSO 4 ·7H 2 O 0.5g，K 2 HPO 4 1g, naCl 0.5g, alkaline lignin 5g, agar powder 22g, distilled water 1L to constant volume, 121 ℃, sterilization for 30min.
LB culture medium: 10.0g of peptone, 5.0g of yeast extract powder, 10.0g of NaCl, 20.0g of agar and 1L of distilled water for constant volume, adjusting the pH to 7.0-7.4, sterilizing at 121 ℃ for 30min.
Aniline blue culture medium: dissolving 0.1g of aniline blue in 10mL of distilled water to obtain aniline blue dye solution; then 10.0g of yeast extract powder, 20.0g of glucose, 15.0g of agar and 990mL of distilled water are taken to prepare a culture medium, the culture medium is sterilized, aniline blue dye solution is absorbed by a disposable medical syringe, a sterile filter is arranged, the aniline blue dye solution is injected into the sterilized culture medium, and the aniline blue dye solution is mixed uniformly in a super clean bench when being hot.
Sodium carboxymethyl cellulose culture 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 powder 0.5g, peptone 2.5g, agar 15.0g, distilled water constant volume to 1L, pH 7.0-7.2, and autoclaving at 121 ℃ for 30min.
The selected Bacillus siamensis (STQ-LIG) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan university and the preservation date of 2022 years, 7 and 25 months, and the preservation number is M20221169.
The Siamese Bacillus (Bacillus siamensis) STQ-LIG of the present example can be grown in solid LB medium.
Morphological characteristics and molecular identification are carried out on the screened Siamese Bacillus (Bacillus siamensis) STQ-LIG as follows.
(1) Morphology characteristics of Siamese bacillus STQ-LIG:
siamese Bacillus (Bacillus siamensis) STQ-LIG that this embodiment was screened is milk white, opaque on solid LB culture medium's bacterial colony, and the surface fold, bacterial colony edge are irregular, the perk, and the centre is sunken, and the bacterial colony surface glues thick. Gram-positive bacteria, rod-shaped.
(2) And (3) molecular identification:
1) Genomic DNA extraction
Inoculating Siamese Bacillus (Bacillus simensis) STQ-LIG strain into a liquid LB culture medium, and performing shaking culture at 28 ℃ for 48 hours to obtain bacterial suspension. And (3) centrifuging the bacterial suspension, washing the bacterial suspension by using sterile water after removing a supernatant, centrifuging again, removing the supernatant to obtain pure bacteria, and then extracting the genome DNA of the bacteria according to the instructions of a genome DNA purification kit (Promega).
2) 16s rDNA PCR amplification
The target gene fragment is obtained by amplification with 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 |
| Is totaled | 25 |
The reaction conditions are as follows: pre-denaturation at 96 ℃ for 5min; 20sec at 96 ℃, 30sec at 62 ℃ and 30sec at 72 ℃ and the cycle number is 35; finally, the resultant was extended at 72 ℃ for 10min and stored at 4 ℃.
3) PCR product detection and purification
3 mul PCR product was detected by 1.0% agarose gel electrophoresis and the band property was observed.
The PCR product purification is carried out according to the operation flow of the magnetic bead purification standard in GB/T40171-2021 general rules for DNA extraction and purification kit by magnetic bead method.
The obtained sequencing sequence was subjected to BLAST (National Center for Biotechnology Information http:// www.ncbi.nlm/nih. Gov ]) homology comparison with nucleic acid data in GenBank, and the result showed that the strain STQ-LIG had 99.86% homology with Bacillus siamensis KCTC 13613, and a phylogenetic tree was constructed using the software MOLECULAR EVOLUTIONARY GENETIC ANALYSIS software (MEGA 5.0), as shown in FIG. 1, and could constitute a stable EVOLUTIONARY branch with Bacillus siamensis KCTC 13613 sequence, and thus, it was named Bacillus siamensis STQ-LIG.
Example 2: sterilized rice straws are subjected to micro-storage by utilizing Siamese Bacillus (Bacillus simensis) STQ-LIG screened in the embodiment 1, and the degradation effect on lignin and cellulose is investigated. The method comprises the following specific steps:
1. culturing 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, adjusting the pH value to 7.0-7.4, and sterilizing at 121 ℃ for 20min.
Selecting a single Siamese Bacillus (Bacillus simensis) STQ-LIG colony after purification, inoculating the single Siamese Bacillus (Bacillus simensis) STQ-LIG colony into a liquid LB culture medium, and culturing for 48 hours on a constant temperature shaking table at 28 ℃ at 200rpm/min to obtain Siamese Bacillus (Bacillus simensis) STQ-LIG bacterial liquid for later use; the bacterial concentration in the bacterial liquid is about 1 × 10 9 cfu/ml。
2. Weighing 0.6g of sterilized rice straw powder in a centrifuge tube, sterilizing 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 comprises: KH (Perkin Elmer) 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 to constant volume, pH value to 7.0-7.4,121 deg.C, sterilizing for 30min. Respectively inoculating 0.4mL of Siamese Bacillus (Bacillus simensis) STQ-LIG bacterial liquid (1 percent of inoculation amount) into a centrifugal tube, obliquely placing the centrifugal tube after inoculation into a shaking table, and rotating for micro-storage under the conditions that the rotating speed is 180rmp and the temperature is 28 ℃; setting for each bacterium3 replicates were performed while a white control group without the addition of Siamese Bacillus (Bacillus siamensis) STQ-LIG was set up. And after 2 weeks of culture, taking out the sample, centrifuging, removing the supernatant, filtering to obtain a fermented sample, weighing the fermented sample, and calculating the loss rate of the straws. And drying the sample obtained after fermentation, measuring the contents of NDF, ADF and ADL of the sample after fermentation, and calculating the degradation rates of the NDF, the ADF and the ADL after fermentation.
The results show that: after 2 weeks of liquid fermentation treatment of rice straws by using Siamese Bacillus (Bacillus siamensis) STQ-LIG bacteria, compared with a blank control group, the content of Neutral Detergent Fiber (NDF), acidic Detergent Fiber (ADF) and Acidic Detergent Lignin (ADL) is reduced by using Siamese Bacillus (Bacillus siamensis) STQ-LIG bacteria liquid to treat the rice straws, the degradation rate of the NDF, the ADF and the ADL is obviously increased, the degradation rate of the NDF, the degradation rate of the ADF and the degradation rate of the ADL are respectively 7.82 percent, 6.21 percent and 4.35 percent in the control group, and the degradation rate of the bacteria liquid treatment group is respectively 18.93 percent, 6.38 percent and 20.22 percent as shown in Table 2.
TABLE 2 degradation of sterilized Rice straw Lignin and cellulose by Bacillus siamensis STQ-LIG bacteria (Dry substance basis)
Example 3: the Siamese Bacillus (Bacillus simensis) STQ-LIG screened in the embodiment 1 is used for micro-storing the unsterilized rice straws, and the degradation effect on lignin and cellulose is investigated. The method comprises the following specific steps:
1. culturing a Siamese Bacillus (Bacillus simensis) STQ-LIG strain:
liquid LB medium: 5g of yeast extract powder, 10g of peptone and 10g of NaCl, adding water to 1000mL, adjusting the pH value to 7.0-7.4, and sterilizing at 121 ℃ for 20min.
Selecting and inoculating a single colony of purified Siamese Bacillus (Bacillus siemensis) STQ-LIG into a liquid LB culture medium, and culturing 4 on a constant temperature shaking table at 28 ℃ at 200rpm/minObtaining Siamese Bacillus (Bacillus siamensis) STQ-LIG bacterial liquid for later use after 8 h; the bacterial concentration in the bacterial liquid is about 1 × 10 9 cfu/ml。
2. Weighing 50g of rice straws in a fermentation bag, adding 2.5mL of Siamese Bacillus (Bacillus simensis) STQ-LIG bacterial liquid and 90mL of distilled water, keeping the water content of the rice straws within the range of 62-66%, vacuumizing, then sealing the fermentation bag, repeating the treatment for three times, weighing the sample after 6 weeks, and determining the influence of microbial liquid storage on chemical components of the rice straws.
The results show that: after the non-sterilized rice straws are fermented by using Bacillus siamensis (STQ-LIG) bacteria liquid for 6 weeks, compared with a blank control group, the content of acid washing lignin, neutral washing fiber and acid washing fiber of the rice straws is respectively reduced by 20.93 percent, 1.82 percent and 3.72 percent in a bacteria liquid treatment group; the degradation rates of the acidic washing lignin and the neutral washing fiber are respectively 2.35 times and 9.68 times of those of the control group, as shown in table 3; the fermentation product increased the content of volatile fatty acids such as acetic acid, propionic acid and butyric acid compared to the control, as shown in table 4; the rice straw dry matter digestibility in vitro was increased by 12.28%, 10.82%, 19.00% and 13.84% at 12, 24, 48 and 72 hours as shown in table 5, and the fiber crystallinity was decreased as shown in fig. 2 by analyzing the XRD spectrum of the fiber by X-ray diffraction as shown in fig. 2.
TABLE 3 degradation of lignin and cellulose in unsterilized rice straw by Bacillus siamensis STQ-LIG bacteria (Dry matter basis)
| Detecting items | Blank control group | Bacterial liquid treatment group |
| Mass% of NDF | 75.46 | 74.09 |
| ADF percent by mass (%) | 51.68 | 49.76 |
| ADL Mass% (percentage) | 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 |
| Ammoniacal nitrogen g/kg | 1.16 | 1.10 |
| Lactic acid g/kg | Undetected | Not detected out |
| 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 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 strain of lignin degrading bacteria is characterized in that the lignin degrading bacteria is Bacillus siamensis STQ-LIG which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan university and the preservation date of 2022 year 7 and 25 days, and the preservation number is M20221169.
2. The use of a strain of lignin-degrading bacteria according to claim 1, characterized in that the use is rice straw micro-storage using Bacillus siamensis (STQ-LIG).
3. The application of the lignin degrading bacteria of claim 2, wherein the lignin degrading bacteria is used for the micro-storage of rice straws, and the method comprises the following steps:
adding the lignin-degrading bacterial liquid into the rice straws, and fermenting at the temperature of 20-30 ℃ to finish the micro-storage of the rice straws.
4. The use of the lignin-degrading bacterium according to claim 3, wherein the concentration of Bacillus siamensis STQ-LIG in the lignin-degrading bacterium solution is 1 x 10 9 ～5×10 9 cfu/mL, wherein the additive amount of the lignin degrading bacteria liquid is 1-5% of the dry weight of the rice straw.
5. The application of the lignin degrading bacterium 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 ligninolytic bacteria according to claim 3 or 4, wherein the fermentation is liquid fermentation or solid fermentation, and the fermentation conditions are anaerobic.
7. The application of the lignin-degrading bacterium according to claim 3 or 4, characterized in that the water content of the rice straw is controlled to be 60-70% by mass under the condition of solid-state fermentation of the rice straw.
8. The use of a strain of lignin-degrading bacteria according to claim 3 or 4, characterized in that the time of the solid state fermentation of rice straw is 5-6 weeks.
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| CN116790423A (en) * | 2023-05-18 | 2023-09-22 | 中国科学院东北地理与农业生态研究所 | Lactobacillus brevis and application thereof in corn or rice straw micro-storage |
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2022
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| CN111925963A (en) * | 2020-08-21 | 2020-11-13 | 四川农业大学 | Application of Siamese bacillus B11 in prevention and/or treatment of cotton bamboo blast tip disease |
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| TIMOTHY D. H. BUGG 等: "Pathways for degradation of lignin in bacteria and fungi", 《NAT. PROD. REP.》, pages 1883 - 1896 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116790423A (en) * | 2023-05-18 | 2023-09-22 | 中国科学院东北地理与农业生态研究所 | Lactobacillus brevis and application thereof in corn or rice straw micro-storage |
| CN116790423B (en) * | 2023-05-18 | 2023-11-28 | 中国科学院东北地理与农业生态研究所 | Lactobacillus brevis and application thereof in corn or rice straw micro-storage |
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| CN115418337B (en) | 2023-06-13 |
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