CN114560726A - Method for promoting efficient utilization of soil organic waste by virtue of earthworm and cellulose decomposition bacteria - Google Patents
Method for promoting efficient utilization of soil organic waste by virtue of earthworm and cellulose decomposition bacteria Download PDFInfo
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- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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Abstract
The invention discloses a method for promoting high-efficiency utilization of soil organic wastes by earthworm in cooperation with cellulose decomposition bacteria, which comprises the following steps: screening cellulolytic bacteria; after the agricultural organic waste is subjected to stack retting treatment, taking a mixture of the agricultural organic waste and soil as a matrix; cellulose decomposing bacteria and earthworms are inoculated into the matrix. By inoculating the earthworms and the cellulose decomposition bacteria, the microbial community structure in the soil micro-domain environment can be changed, the activity of the cellulose decomposition bacteria is excited, and the degradation of the agricultural organic waste is effectively promoted. On one hand, the digestion of the earthworms is helpful for improving the availability of cellulose in agricultural organic waste, and can induce microorganisms to generate cellulase, thereby improving the activity of the cellulase. On the other hand, mucus and excrement of the earthworms can activate the metabolism of microorganisms, and have certain influence on the production of cellulase by the microorganisms.
Description
Technical Field
The invention relates to the technical field of agricultural waste treatment, in particular to a method for promoting high-efficiency utilization of soil organic waste by cooperating earthworm with cellulose decomposition bacteria.
Background
China is the largest agricultural production country in the world, the yield of straws is the top of the world, and the amount of various crop straws generated by the planting industry in China is up to 7 hundred million tons every year according to statistics. The agricultural wastes such as straws and the like are main byproducts of agricultural production, contain a large amount of organic carbon and nutrient elements such as nitrogen, phosphorus, potassium and the like which are necessary for the growth of plants, and if the agricultural wastes are recycled, not only can the ecological environment be protected, but also the soil fertility can be improved, and the sustainable development of the agricultural production can be promoted. However, the utilization rate of agricultural wastes such as straws in China is very low, and the average utilization rate is only 32 percent and is far lower than that of developed countries such as United kingdom and America. A large amount of agricultural wastes are randomly burned or discarded, so that serious atmospheric pollution is caused, the traffic accident incidence is increased, the morbidity of respiratory diseases is increased, and the human health is seriously harmed. The organic materials such as agricultural wastes and the like have poor recycling effect and low decomposition rate, and the application of the organic fertilization technology in agricultural production is severely limited. The key reason for the low utilization rate of agricultural wastes is that the agricultural wastes contain 40-50% of cellulose, which is a compound with a complex structure and difficult degradation. How to increase the utilization rate of agricultural organic wastes such as straws and the like and improve the capability of the agricultural organic wastes to compensate soil nutrients is one of the main problems in agricultural production in China.
CN 108456017A discloses a method for treating general waste by using earthworms, plants and probiotics. Preparing a waste treatment mixture, crushing the waste, uniformly placing the mixture in the waste, stacking the waste after mixing and stirring, uniformly spraying probiotics on the outer surface of a stacking area, placing the treated waste in a fermentation tank again, uniformly adding earthworms into the fermentation tank, standing for 15-18 days at 15-30 ℃, mixing and stirring the waste in the fermentation tank every 3 days in the process, secreting special enzyme, and decomposing the waste again by the obtained special enzyme to generate a nutritional mixed material.
CN 106342434A discloses a method for promoting fast-growing forest trees to grow by improving soil nitrogen nutrients by using an earthworm agent. The application of the pig manure, the biogas slurry, the earthworms and the microbial inoculum can not only quickly improve the effective nitrogen content of soil and the stability of nitrogen fertility, but also promote the growth of fast growing wood, respectively improve the base diameter, the plant height and the crown width by 1.7 times, 1.3 times and 5.1 times relative to CK, and eliminate the pollution of livestock and poultry manure. The research of the invention shows that the application of the earthworm and the microbial inoculum can improve the decomposition rate of the pig manure in the soil and increase the nitrogen content of the soil; secondly, some nitrogen in the air which cannot be utilized by plants is converted into available nitrogen; and thirdly, the earthworms and the microbial inoculum contain carbon and nitrogen nutrients, and the combined application of the earthworms and the microbial inoculum can improve the nitrogen content of the soil.
CN 105814997A discloses a method for enhancing soil carbon sequestration capacity by using an earthworm agent. The method comprises the step of applying pig manure, biogas slurry, a composite microbial inoculum and mixed earthworms in seasons within the range of 1m2 surrounding each forest in the fast-growing forest land and within the depth range of 0-60cm of soil. Researches show that on one hand, the earthworms can promote the degradation and transformation of the pig manure and accelerate the humification process of the pig manure, and the combined application of the microbial inoculum and the earthworms has obvious and rapid synergistic effect on the accumulation of organic matters in soil. On the other hand, half of the organic matter accumulated is stabilized organic carbon. In conclusion, the pig manure, the biogas slurry, the earthworms and the microbial inoculum are applied together, so that the quantity and the quality of soil carbon fixation can be rapidly improved, the soil fertility can be improved, the growth of fast-growing forest trees can be promoted, a large amount of manure waste can be consumed, and the win-win effect of ecological benefit, environmental benefit and economic benefit can be realized.
At present, the research and development of the technology at home and abroad mostly concentrate on improving the soil fertility by using earthworms to cooperate with microbial inoculum, and almost no method for promoting the high-efficiency utilization of agricultural organic wastes in the tea garden soil by combining earthworms with cellulose decomposition bacteria exists. In addition, the prior art generally adopts artificially-cultured Eisenia foetida (Eisenia foetida) combined with the composite probiotic preparation, and has the defects of slow effect, complex formula and the like.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for promoting the efficient utilization of soil organic wastes by using earthworms and cellulose decomposing bacteria.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for promoting the efficient utilization of soil organic wastes by the cooperation of the earthworms and the cellulose decomposition bacteria comprises the following steps:
(1) screening cellulose decomposition bacteria, wherein the cellulose decomposition bacteria are taken from tea garden soil, and the specific steps of screening the cellulose decomposition bacteria are as follows;
A) enriching cellulolytic bacteria;
B) primary screening of cellulose decomposition bacteria: primarily screening the cellulose decomposition bacteria by adopting a dilution coating flat plate method and a Congo red dyeing method;
C) re-screening cellulose decomposition bacteria: re-screening the cellulolytic bacteria by an ELISA kit and a filter paper degradation rate;
D) identifying cellulolytic bacteria;
(2) after the agricultural organic waste is subjected to stack retting treatment, taking a mixture of the agricultural organic waste and soil as a matrix;
(3) cellulose decomposing bacteria and earthworms are inoculated into the matrix.
As a further improvement of the invention: the specific steps of the enrichment of cellulolytic bacteria in step a) include: burying rice straws and tea tree withered branches and fallen leaves in tea garden soil, taking out the rice straws and the soil around the rice straws after half a year to prepare an extracting solution, adding the extracting solution into a sterilization selective culture medium, and putting the selective culture medium into a constant-temperature oscillation incubator for culture;
as a further improvement of the invention: the preliminary screening of the cellulolytic bacteria in the step B) comprises the following specific steps: diluting the enriched cellulolytic bacteria with a bacterial liquid, coating the bacterial liquid on an identification medium plate by adopting a dilution coating plate method, putting the identification medium plate into a constant-temperature incubator for culture, after the bacterial grows out, selecting bacterial colonies with obvious colony morphology difference, and repeatedly scribing and inoculating the bacterial colonies on the identification medium plate until single bacterial colonies are obtained after purification; primarily screening the capacity of the purified single colony for degrading cellulose of the strain by using a Congo red dyeing method to obtain the strain with higher cellulase activity;
as a further improvement of the invention: the specific steps of the secondary screening of the cellulolytic bacteria in the step C) comprise: selecting bacteria with better primary screening results to perform a cellulase activity verification experiment, and comparing the activities of exoglucanase, endoglucanase and beta-glucosidase by using an ELISA kit to obtain bacteria with higher cellulase activity; performing a filter paper verification test on the bacteria with higher cellulase activity, selecting a strain with higher cellulase activity from an identification culture medium flat plate, culturing the strain in an LB liquid culture medium at constant temperature to activate the strain, absorbing the strain liquid into a triangular flask filled with the filter paper liquid culture medium, sealing the bottleneck by using a sterilization film, and culturing the triangular flask in a constant-temperature oscillator; after the culture is finished, drying the culture together with the triangular flask to constant weight, and calculating the degradation rate of the filter paper; re-screening the capacity of the strain for degrading cellulose by the filter paper degradation rate to obtain the strain with higher cellulase activity;
as a further improvement of the invention: the specific steps for identifying the cellulolytic bacteria in step D) comprise: and sequencing the high-efficiency cellulolytic bacteria with higher enzyme activity in the re-screening result, performing homology comparison analysis on the obtained sequence result and the sequence result of an NCBI website, selecting a sequence with higher homology, and constructing a genetic system evolutionary tree by using a Neighbor-Joining method in MEGA7 software.
As a further improvement of the invention: the proportion of the agricultural organic waste to the soil in the step (2) is 1: 14-1: and 15, after fully mixing the agricultural organic waste with the soil, adjusting the C/N to be 18: 1-20: 1.
as a further improvement of the invention: the agricultural organic waste in the step (2) is 11.5g, the soil is 168.5g, and after the agricultural organic waste and the soil are fully mixed, the C/N is adjusted to be 20: 1.
as a further improvement of the invention: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: inoculating several earthworms into the matrix, and inoculating cellulose decomposing bacteria after 14 days.
As a further improvement of the invention: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: the matrix is inoculated with cellulose decomposing bacteria, and a plurality of earthworms are inoculated after 14 days.
As a further improvement of the invention: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: inoculating a plurality of earthworms and cellulose decomposition bacteria into the matrix at the same time.
As a further improvement of the invention: the earthworm is wild earthworm.
Compared with the prior art, the invention has the beneficial effects that:
the earthworm and the cellulose decomposition bacteria can excite the activity of the cellulose decomposition bacteria by changing the microbial community structure in the soil micro-domain environment, thereby effectively promoting the degradation of the agricultural organic waste. On one hand, the digestion of the earthworms is helpful for improving the availability of cellulose in agricultural organic waste, and can induce microorganisms to generate cellulase, thereby improving the activity of the cellulase. In another aspect, mucus and feces from earthworms can activate microbial metabolism.
Drawings
FIG. 1 is a schematic diagram of exoglucanase activity of different species.
FIG. 2 is a schematic diagram showing endoglucanase activities of different strains.
FIG. 3 is a diagram showing the activity of beta-glucosidase in different strains.
FIG. 4 is a graph showing the effect of different bacteria on degrading filter paper.
FIG. 5 is a phylogenetic tree of strain No. 14.
FIG. 6 is a phylogenetic tree of strain No. 15.
FIG. 7 is a phylogenetic tree of strain No. 16.
FIG. 8 is a phylogenetic tree of strain No. 26.
FIG. 9 is a graph showing the effect of different treatments on soil exoglucanase activity.
FIG. 10 is a graph showing the effect of different treatments on the activity of soil endoglucanases.
FIG. 11 is a graph showing the effect of different treatments on soil beta-glucosidase activity.
Detailed Description
In this document, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. The invention will now be further described with reference to the accompanying description and examples:
the first embodiment is as follows:
the method for promoting the efficient utilization of soil organic wastes by the cooperation of the earthworms and the cellulose decomposition bacteria comprises the following steps:
(1) screening cellulose decomposition bacteria, wherein the cellulose decomposition bacteria are taken from tea garden soil, and the specific steps of screening the cellulose decomposition bacteria are as follows;
A) enriching cellulose decomposition bacteria;
B) primary screening of cellulose decomposition bacteria: primarily screening the cellulose decomposition bacteria by adopting a dilution coating flat plate method and a Congo red dyeing method;
C) re-screening cellulose decomposition bacteria: re-screening the cellulolytic bacteria by an ELISA kit and a filter paper degradation rate;
D) identifying cellulolytic bacteria;
(2) after the agricultural organic waste is subjected to stack retting treatment, taking a mixture of the agricultural organic waste and soil as a matrix;
(3) cellulose decomposing bacteria and earthworms are inoculated into the matrix.
The agricultural organic waste in the step (2) is straw.
The specific steps of the enrichment of cellulolytic bacteria in step a) include: burying rice straw and tea tree dry branches and fallen leaves in tea garden soil, taking out rice straw and surrounding soil after half a year to prepare 1:1 extract, adding 10mL of extract into 150mL of sterilization selective culture medium, placing the selective culture medium into a constant temperature oscillation incubator for culturing for 48 hours at the rotation speed of 200 r.min-1The temperature was 36 ℃.
The specific steps of the dilution coating flat plate method in the step B) comprise: diluting the enriched cellulolytic bacteria with bacteria liquid, wherein the dilution step is carried out according to the conventional bacteria liquid dilution method, and the dilution degree is 10-2、10-3、10-4、10-5Coating 0.2mL of each sample on an identification medium plate, coating 3 samples on each gradient, and putting the samples into a constant-temperature incubator with a constant temperature of 36 ℃ for culturing for 60 hours; after the colonies grow out, selecting the colonies with obvious colony morphology difference, repeatedly streaking and inoculating the colonies on an identification medium plate until single colonies are obtained through purification, then transferring the single colonies onto corresponding agar slopes, and storing the single colonies for later use at 4 ℃.
The Congo red dyeing method in the step B) comprises the following specific steps: primarily screening the purified strain for the capacity of degrading cellulose of the strain by using a Congo red dyeing method to obtain the strain with higher cellulase activity;
judging the activity of the cellulase according to the size of a hydrolysis ring generated by the strain on an identification medium flat plate;
in this example, a part of the strains generate hydrolysis rings on a carboxymethyl cellulose-congo red plate, and the larger the cellulose hydrolysis ring is, the more strong the cellulase activity is, the stronger the capability of the strains to degrade cellulose is, and the size of the generated hydrolysis ring is shown in table 1; wherein, the average diameter of the hydrolysis ring of the No. 36 strain is the largest and reaches 5.57cm, the hydrolysis ring generated by the No. 5 strain is the smallest, and the average diameter is only 0.33 cm. In addition, among the screened species, 9 species produced hydrolysis rings with an average diameter of greater than 4 cm.
| Strain numbering | Average diameter (cm) of transparent ring | Strain numbering | Average diameter (cm) of transparent ring |
| 1 | 0.53±0.14 | 22 | 4.33±0.34 |
| 2 | 0.62±0.16 | 23 | 1.8±0.92 |
| 3 | 2.2±2.33 | 24 | 3.47±0.33 |
| 5 | 0.33±0.21 | 25 | 3.43±0.33 |
| 6 | 2.73±1.28 | 26 | 3.57±0.29 |
| 7 | 0.47±0.05 | 28 | 2.73±1.08 |
| 8 | 0.47±0.06 | 29 | 4.22±0.06 |
| 9 | 0.45±0.07 | 31 | 4.02±0.13 |
| 10 | 0.57±0.09 | 32 | 3.97±0.16 |
| 11 | 0.42±0.06 | 34 | 3.77±0.16 |
| 12 | 0.5±0 | 35 | 4.28±0.16 |
| 13 | 0.5±0.08 | 36 | 5.57±0.59 |
| 14 | 2.82±0.2 | 37 | 2.97±1.11 |
| 15 | 2.85±0.05 | 38 | 3.97±0.05 |
| 16 | 4.9±0 | 39 | 3.28±1.27 |
| 17 | 3.87±0.17 | 42 | 2.15±0 |
| 19 | 4.13±1.33 | 43 | 2.2±0 |
| 20 | 4.9±0.14 | 44 | 2.27±0.16 |
| 21 | 4.17±1.04 |
TABLE 1 preliminary screening results for cellulolytic bacteria
The specific steps of the secondary screening of the cellulolytic bacteria in the step C) comprise: selecting bacteria with better primary screening results to perform a cellulase activity verification experiment, and comparing the activities of exoglucanase, endoglucanase and beta-glucosidase by using an ELISA kit to obtain bacteria with higher cellulase activity; performing a filter paper verification test on the bacteria with higher cellulase activity, selecting a single bacterial colony from an identification culture medium plate, placing the single bacterial colony in an LB liquid culture medium, and culturing at the constant temperature of 37 ℃ for 18 hours to activate the bacterial strain; 2.5mL of bacterial liquid is sucked into a 150mL triangular flask filled with 50mL of corresponding filter paper liquid culture medium, and the bottle mouth is sealed by a sterilization film; placing the triangular flask in a constant temperature oscillator at 36 deg.C for 120 r.min-1The rotation speed of (3) and culturing for 10 days; after the culture is finished, drying the culture together with a triangular flask to constant weight, wherein the constant weight is determined by that the continuous weighing change is less than 0.05%, and each strain is set to be 3 times; measuring the dry mass m1 of the dried filter paper by a subtraction method, taking the dry mass m2 of the dried filter paper in a culture medium without inoculated bacterial liquid as a blank control, and calculating the degradation rate of the filter paper by the following formula:
re-screening the capacity of the strain for degrading cellulose by the filter paper degradation rate to obtain the strain with higher cellulase activity;
in this embodiment, 11 kinds of bacteria with better primary screening results are selected for further cellulase activity verification experiments, and the activities of exoglucanase, endoglucanase and β -glucosidase are compared by an ELISA kit, and the experimental results are shown in fig. 1 to 3, from which it can be seen that the cellulase activities of bacteria No. 14, 15, 16 and 26 are higher;
meanwhile, the filter paper verification test is carried out on the 4 bacteria with higher cellulase activity, the experimental result is shown in figure 4, and the figure shows that the degradation effect of the No. 26 bacteria is obvious, and the degradation rate reaches 22.04%.
The specific steps for identifying the cellulolytic bacteria in step D) comprise: sequencing the high-efficiency cellulolytic bacteria with higher enzyme activity in the re-screening result, carrying out homology comparison analysis on the obtained sequence result and the sequence result of the NCBI website, selecting a sequence with higher homology, and constructing a gene system evolutionary tree by using a Neighbor-Joining method in MEGA7 software;
and (3) taking the strains with higher enzyme activity in the re-screening result as high-efficiency cellulose decomposition bacteria, and extracting the DNA of the high-efficiency cellulose decomposition bacteria, wherein the DNA of the strains is extracted by using a Tiangen DNA extraction kit. The 16S rDNA sequence of the strain was PCR amplified using the total DNA as template and the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The 50. mu.L PCR reaction system contained 2 XT 5 Mix 25. mu.L, 1. mu.L of bacterial suspension, 4. mu.L each of primers 27-F and 1492-R, and ddH2O 16. mu.L. The PCR reaction program is: pre-denaturation at 94 ℃ for 10 min; denaturation at 98 ℃ for 10 seconds, renaturation at 55 ℃ for 10 seconds, extension at 72 ℃ for 20 seconds, and 30 cycles; final extension at 72 ℃ for 5 min. After the PCR product is successfully identified by agarose gel electrophoresis, gel recovery and sequencing are carried out; purifying PCR products by using a DNA gel recovery kit, and sequencing amplified products by sanger;
in this embodiment, bacteria 14, bacteria 15, bacteria 16, and bacteria 26 with better rescreening results are sequenced, the obtained sequence results are compared with the sequence results of the NCBI website for homology analysis, a sequence with higher homology is selected, and a genetic system evolutionary tree is constructed by using a Neighbor-Joining method in MEGA7 software, wherein the experimental results are shown in fig. 5 to 8; as can be seen from the figure, it was confirmed that bacterium No. 14 was Brevibacillus brevis (Brevibacillus parabrevis), bacterium No. 15 was Brevundimonas diminuta (Brevundimonas diminuta), bacterium No. 16 was Ochrobactrum intermedium (Ochrobactrum intermedium), and bacterium No. 26 was Bacillus proteolicus (Bacillus proteoliticus).
The earthworm is wild earthworm. The cellulose decomposition bacteria are high-efficiency cellulose decomposition bacteria screened from soil of a typical tea garden in Guangdong province.
Example two (E treatment):
screening high-efficiency cellulose decomposing bacteria from tea garden soil, composting the straws, and adjusting C/N to 20: 1, only 4 earthworms are inoculated and cultured for 28 days.
Example three (B treatment):
screening high-efficiency cellulose decomposing bacteria from tea garden soil, composting the straws, and adjusting C/N to 20: 1, inoculating only cellulolytic bacteria with the inoculum size of 8.25 × 106cfu, cultured for 28 days.
Example four (EB treatment):
screening high-efficiency cellulose decomposing bacteria from tea garden soil, composting the straws, and adjusting C/N to 20: 1, inoculating 4 earthworms, inoculating cellulose decomposing bacteria after 14 days, wherein the inoculation number is 8.25 multiplied by 106cfu, further cultured for 14 days.
Example five (BE treatment):
screening high-efficiency cellulose decomposing bacteria from tea garden soil, composting the straws, and adjusting C/N to 20: 1, inoculating cellulolytic bacteria with the inoculum size of 8.25 × 106cfu, 4 earthworms were inoculated 14 days later and cultured for another 14 days.
Example six (E + B treatment):
screening high-efficiency cellulose decomposing bacteria from tea garden soil, composting the straws, and adjusting C/N to 20: 1, simultaneously inoculating 4 earthworms and 8.25X 10 earthworms6cfu cellulolytic bacteria, cultured for 28 days.
Blank control (CK treatment):
after the straw is subjected to composting treatment, the C/N is adjusted to be 20 by taking a mixture of 11.5g of straw and 168.5g of soil as a matrix: 1, culturing for 28 days.
Wherein the Lumbricus is Amynthas cortex (Amynthas corticis) and is obtained from soil in Ender tea garden, and the weight of 4 Lumbricus is 2.50 + -0.10 g.
After the culture is finished, determining the diversity of soil microbial communities treated differently and the influence of different treatments on the activities of soil exoglucanase, endoglucanase and beta-glucosidase; wherein the soil microbial community diversity is shown in the following table 2:
| treatment of | chao1 index | Shannon index | Simpson index |
| CK | 3583.39±30.43bc | 8.80±0.06b | 0.99±ab |
| B | 3927.57±32.59a | 9.19±0.03a | 1.00±a |
| E | 3183.87±68.65d | 7.99±0.14c | 0.99±cd |
| BE | 3648.38±75.64b | 8.84±0.14ab | 0.99±ab |
| EB | 3403.56±46.35c | 8.09±0.15c | 0.98±d |
| E+B | 3418.13±82.16c | 8.29±0.14c | 0.99±bc |
TABLE 2 soil microbial community diversity index Table
The effects of different treatments on the activities of soil exoglucanase, endoglucanase and beta-glucosidase were compared by ELISA kits, and the experimental results are shown in fig. 9 to 11;
from the experimental results, it was found that the BE treatment, the EB treatment and the E + B treatment have a promoting effect on the activity of cellulolytic bacteria compared with the E treatment and the CK treatment, and the promoting effect of the BE treatment on the activity of cellulolytic bacteria is the most preferable;
experimental results show that the earthworms and the cellulose decomposition bacteria stimulate the activity of the cellulose decomposition bacteria by changing the microbial community structure in the soil micro-domain environment, and effectively promote the degradation of the straws. On one hand, the digestion of the earthworms is beneficial to improving the availability of cellulose in the straws, and can induce microorganisms to generate cellulase, thereby improving the activity of the cellulase. On the other hand, mucus and excrement of the earthworms can activate the metabolism of microorganisms, and have certain influence on the production of cellulase by the microorganisms.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; these modifications and substitutions do not cause the essence of the corresponding technical solution to depart from the scope of the technical solution of the embodiments of the present invention, and are intended to be covered by the claims and the specification of the present invention.
Claims (10)
1. The method for promoting the efficient utilization of soil organic wastes by the cooperation of the earthworms and the cellulose decomposition bacteria is characterized by comprising the following steps: the method comprises the following steps:
(1) screening cellulose decomposition bacteria, wherein the cellulose decomposition bacteria are taken from tea garden soil, and the specific steps of screening the cellulose decomposition bacteria are as follows;
A) enriching cellulolytic bacteria;
B) primary screening of cellulose decomposition bacteria: primarily screening the cellulose decomposition bacteria by adopting a dilution coating flat plate method and a Congo red dyeing method;
C) re-screening cellulose decomposing bacteria: re-screening the cellulolytic bacteria by an ELISA kit and a filter paper degradation rate;
D) identifying cellulolytic bacteria;
(2) after the agricultural organic waste is subjected to stack retting treatment, taking a mixture of the agricultural organic waste and soil as a matrix;
(3) cellulose decomposing bacteria and earthworms are inoculated into the matrix.
2. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 1, wherein the method comprises the following steps: the specific steps of the enrichment of cellulolytic bacteria in step a) include: burying rice straw and tea tree dry branches and fallen leaves in tea garden soil, taking out the rice straw and the surrounding soil after half a year, preparing an extracting solution, adding the extracting solution into a sterilization selective culture medium, and putting the selective culture medium into a constant-temperature oscillation culture box for culture.
3. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria as claimed in claim 2, wherein: the preliminary screening of the cellulolytic bacteria in the step B) comprises the following specific steps: diluting the enriched cellulolytic bacteria with a bacterial liquid, coating the bacterial liquid on an identification medium plate by adopting a dilution coating plate method, putting the identification medium plate into a constant-temperature incubator for culture, after the bacterial grows out, selecting bacterial colonies with obvious colony morphology difference, and repeatedly scribing and inoculating the bacterial colonies on the identification medium plate until single bacterial colonies are obtained after purification; and primarily screening the cellulose degrading capacity of the purified single colony by using a Congo red dyeing method to obtain the strain with higher cellulase activity.
4. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 3, wherein the method comprises the following steps: the specific steps of re-screening the cellulolytic bacteria in the step C) comprise: selecting bacteria with better primary screening results to perform a cellulase activity verification experiment, and comparing the activities of exoglucanase, endoglucanase and beta-glucosidase by using an ELISA kit to obtain bacteria with higher cellulase activity; performing a filter paper verification test on the bacteria with higher cellulase activity, selecting a strain with higher cellulase activity from an identification culture medium flat plate, culturing the strain in an LB liquid culture medium at constant temperature to activate the strain, absorbing the strain liquid into a triangular flask filled with the filter paper liquid culture medium, sealing the bottleneck by using a sterilization film, and culturing the triangular flask in a constant-temperature oscillator; after the culture is finished, drying the culture together with the triangular flask to constant weight, and calculating the degradation rate of the filter paper; and re-screening the capacity of the strain for degrading cellulose by the filter paper degradation rate to obtain the strain with higher cellulase activity.
5. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 4, wherein the method comprises the following steps: the specific steps for identifying the cellulolytic bacteria in step D) comprise: and sequencing the high-efficiency cellulolytic bacteria with higher enzyme activity in the re-screening result, carrying out homology comparison analysis on the obtained sequence result and the sequence result of the NCBI website, selecting a sequence with higher homology, and constructing a gene system evolutionary tree by using a Neighbor-Joining method in MEGA7 software.
6. The method for promoting the efficient utilization of the organic waste in the soil by the earthworm and the cellulolytic bacteria, according to claim 5, wherein the method comprises the following steps: the proportion of the agricultural organic waste to the soil in the step (2) is 1: 14-1: and 15, after fully mixing the agricultural organic waste with the soil, adjusting the C/N to be 18: 1-20: 1.
7. the method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria as claimed in claim 6, wherein: the agricultural organic waste in the step (2) is 11.5g, the soil is 168.5g, and after the agricultural organic waste and the soil are fully mixed, the C/N is adjusted to be 20: 1.
8. the method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 7, wherein the method comprises the following steps: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: inoculating a plurality of earthworms into the matrix, and inoculating cellulose decomposition bacteria after a plurality of days.
9. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 7, wherein the method comprises the following steps: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: the matrix is inoculated with cellulose decomposing bacteria firstly, and a plurality of earthworms are inoculated after a plurality of days.
10. The method for promoting the efficient utilization of soil organic waste by the earthworms cooperating with the cellulolytic bacteria according to claim 7, wherein the method comprises the following steps: the specific steps of inoculating the cellulose-decomposing bacteria and the earthworms into the matrix in the step (3) comprise: simultaneously inoculating a plurality of earthworms and cellulose decomposing bacteria into the matrix.
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| US20190194081A1 (en) * | 2015-12-20 | 2019-06-27 | Envirokure, Incorporated | Nutritional Compositions For Plants And Soils |
| CN111690539A (en) * | 2020-07-07 | 2020-09-22 | 安徽农业大学 | Screening and application of high-efficiency straw cellulose decomposition bacteria |
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| US20190194081A1 (en) * | 2015-12-20 | 2019-06-27 | Envirokure, Incorporated | Nutritional Compositions For Plants And Soils |
| CN106434417A (en) * | 2016-08-01 | 2017-02-22 | 奥为（天津）环保科技有限公司 | High-temperature-resistant cellulase producing bacterium and application thereof |
| CN111690539A (en) * | 2020-07-07 | 2020-09-22 | 安徽农业大学 | Screening and application of high-efficiency straw cellulose decomposition bacteria |
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