CN115161218B - Bacterial strain capable of degrading agricultural dry branch and fallen leaf waste, screening method and application thereof - Google Patents
Bacterial strain capable of degrading agricultural dry branch and fallen leaf waste, screening method and application thereof Download PDFInfo
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Abstract
The invention provides a bacterial strain capable of degrading agricultural dry branch and fallen leaf wastes, a screening method and application thereof, wherein the bacterial strain MDD 2020 belongs toOchrobactrumpseudintermeddium(pseudo-middle pallor) deposited in China general microbiological culture Collection center (CGMCC) with a culture collection number of CGMCC No.22199, 4.about.19.2021. The method for degrading agricultural dead-branch fallen-leaf waste by utilizing the strain MDD 2020 fermentation promotes the decomposition process of dead-branch fallen-leaf compost, provides technical support for popularization and utilization of dead-branch fallen-leaf composting, achieves the purposes of changing waste into valuables, protecting environment and promoting agricultural sustainable development, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of strain biology, and particularly relates to a bacterial strain capable of degrading agricultural branches and leaves of dead branches and fallen leaves, a screening method and application thereof.
Background
Lignin is a complex high molecular compound constituting the xylem of plants, and is the second most abundant plant-derived material next to cellulose. Lignin is an important component of plant cell walls and accounts for about 15% -40% of the dry mass of plants. The annual photosynthetic yield of lignin in nature is about 200 x 10 8 Tons. Because the lignin structure is complex, the lignin-rich agricultural and garden dead branch and fallen leaf wastes are not easy to rot and degrade, and the compost is difficult to return to the fieldMost of the waste water is randomly piled up on the roadside or burned on the spot, so that serious atmospheric pollution is caused, diseases and insect pests in the field are induced in the piling up process, and meanwhile, valuable resources are wasted.
The lignin in the agricultural waste is recycled (composted) as solid organic waste, which is a simple and effective way to solve the problem. Research shows that the garden waste is used as a conditioner of the compost material, and has good regulation effect on the water content, the porosity, the carbon-nitrogen ratio and the like of the compost material. And lignin has a complex structure and high degradation difficulty. The lignin degrading strain under the natural condition is single in species and small in quantity, so that the lignin degrading rate is limited, and the composting progress is hindered. By inoculating lignin degrading bacterial agent, the temperature rise of the pile body can be accelerated, and the decomposition of lignin in the compost substrate can be accelerated, so that the composting period is shortened. The result of the combined composting experiment shows that the composting of the dry branches and fallen leaves is rich and comprehensive in nutrition, and the quantity of germs is harmless, so that the fertilizer is a mature, stable and high-quality organic fertilizer and soil conditioner rich in nutrition. The method can realize the recycling (composting) of solid organic waste by the dead branches and fallen leaves, and is an effective way for solving the harmless and recycling problems. If the corresponding high-efficiency degradation functional strain can be screened and used for the composting production of agricultural dry branch and fallen leaf wastes, the method has good application prospect and practical value.
Disclosure of Invention
In order to overcome the defects in the prior art, the bacterial strain capable of degrading agricultural dry-branch and fallen-leaf wastes and the screening method thereof are provided, and the aim of screening bacteria capable of efficiently decomposing lignin in agricultural dry-branch and fallen-leaf wastes is achieved by the efficient degradation effect of the bacteria on lignin, so that the aim of accelerating composting and decomposing of the dry-branch and fallen-leaf wastes is fulfilled. Therefore, the agricultural dry branch and fallen leaf waste can be recycled in a large scale through composting, and the smooth development of sustainable agriculture is ensured.
In order to solve the technical problems, the invention provides a bacterial strain capable of degrading agricultural branches and leaves of dead branches, which belongs to the genus of pallor, wherein the bacterial strain MDD 2020 belongs to the genus of pseudo-middle pallor, and is deposited in China general microbiological culture Collection center (CGMCC) with a culture collection number of CGMCC No.22199, and the day 19 of 2021.
As another aspect of the present invention, the present invention provides a method for screening MDD 2020 bacterial strains, which comprises subjecting lignin-degrading bacteria to solid enrichment and liquid enrichment with dry branch and fallen leaf powder as the sole carbon source for 30 d two consecutive generations, and performing separation and purification on guaiacol selection medium.
Preferably, the solid enrichment and liquid enrichment use culture mediums which are respectively a solid enrichment culture medium and a liquid enrichment culture medium, and the formula is as follows: the mass volume ratio of the dry branch fallen leaf powder to the water is 2g/ml; liquid enrichment medium: (NH) 4 ） 2 SO 4 0.5g/L，KH 2 PO 4 1g/L，MgSO 4 ·7 g/L H 2 O 0.5 g/L。
Preferably, the strain MDD 2020 is inoculated into an enzyme-producing medium and cultured for 9-21d in a shaking manner.
Preferably, the formulation of the enzyme-producing medium is (/ L): glucose 20 g, ammonium tartrate 0.2 g, pear tree branch powder 1 g, basal medium 100 mL,0.1 mol/L NaAc-HAc buffer (pH=4.5) 100 mL, tween 80.0 g VB 1 1.0 mg。
As another aspect of the present invention, there is provided a medium for screening of MDD 2020 bacterial strain, characterized in that: comprises a solid enrichment culture medium and/or a liquid enrichment culture medium, wherein the formula is as follows: the mass volume ratio of the dry branch fallen leaf powder to the water is 2g/ml; liquid enrichment medium: (NH) 4 ） 2 SO 4 0.5g/L，KH 2 PO 4 1g/L，MgSO 4 ·7H 2 O 0.5 g/L。
As a further aspect of the invention, the invention provides the use of a strain of MDD 2020 bacteria.
The invention has the beneficial effects that:
the invention provides a bacterial strain capable of degrading agricultural branches and leaves of dead and falling waste, wherein the strain MDD 2020 belongs to Ochrobactrum pseudintermeddium (pseudo-middle pallium), is deposited in China academy of sciences microbiological study, china general microbiological culture Collection center, china Committee for culture Collection of microorganisms, has a collection address of Beijing, chaoyang area, north Qinghai, west Lu No. 1, 3, a culture collection number of CGMCC No.22199, and a suggested classification name of Ochrobactrum ciceri. The biological characteristics are as follows: on LB culture medium, at 37 deg.C, the colony is nearly circular, milky white, moist, smooth and slightly convex, single colony has a diameter of about 4mm, gram negative, positive for contact enzyme and oxidase. Indole was negative. The method for degrading agricultural dead-branch fallen-leaf waste by utilizing microbial strain MDD 2020 fermentation promotes the decomposition process of dead-branch fallen-leaf compost, provides technical support for popularization and utilization of dead-branch fallen-leaf composting, achieves the purposes of changing waste into valuables, protecting environment and promoting agricultural sustainable development, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the fading reaction of the bacterial MDD 2020 of the present invention;
FIG. 2 is a phylogenetic tree of bacterial MDD 2020 of the present invention based on 16S rDNA sequence similarity;
FIG. 3 is a graph showing the activity of Lip lignin peroxidase in a crude enzyme broth fermented by the bacterium MDD 2020 of the present invention;
FIG. 4 is a graph showing the activity of MnP manganese peroxidase in a crude enzyme fermentation broth of the bacterial MDD 2020 of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
The culture medium formula and enzyme activity and solid degradation capacity measurement method used in the invention are as follows:
solid enrichment medium: and 20 g of dry branch fallen leaf powder (fallen leaves and broken branches of peach tree covered by the upper layer of peach garden soil), cleaning with distilled water, drying, crushing, sieving with a 60-mesh sieve, and 10 ml of distilled water.
Liquid enrichment medium (/ L): (NH) 4 ） 2 SO 4 0.5g，KH 2 PO 4 1g，MgSO 4 ·7H 2 O 0.5 g。
Guaiacol selection medium (/ L): guaiacol 1 g, KNO 3 2 g，MgSO 4 0.5 g，KH 2 PO 4 1 g，NaCl 1 g，Na 2 HPO 4 1 g, agar 20 g.
LB-guaiacol medium: adding 1 g.L into LB culture medium -1 Guaiacol.
LB-guaiacol medium: adding 1 g.L into LB culture medium -1 Guaiacol.
LB-aniline blue medium: LB medium was added with 0.1 g.L -1 Aniline blue.
Enzyme production medium (/ L): glucose 20 g, ammonium tartrate 0.2 g, dry-branch fallen leaf powder 1 g (fallen leaves and broken branches of peach tree covered by peach garden soil upper layer, distilled water cleaning, oven drying, pulverizing, sieving with 60 mesh sieve) 1 g, basic culture medium 100 mL,0.1 mol/L NaAc-HAc buffer solution (pH=4.5) 100 mL, tween 80.0 g VB 1 1.0 mg。
Basal medium (/ L): k (K) 2 PO 4 20 g ，MnSO 4 5 g，CaCl 2 1 g, trace element liquid 100 mL.
Trace element liquid (/ L): mgSO (MgSO) 4 3.0g，MnSO 4 0.5 g，NaCl 1.0 g，FeSO 4 ·7H 2 SO 4 0.1 g， CoCl 2 0.1 g， CuSO 4 0.1 g， H 3 BO 3 0.01 g， ZnSO 4 ·7H 2 O 0.1 g ， AlK（SO 4 ） 2 ·12H 2 O 0.01 g， Na 2 MoO 4 · 2H 2 O 0.01 g。
Solid culture nutrient solution (L): NH 4 Cl 2.0 g，MgSO 4 ·7H2O 0.5 g，KH 2 PO 4 1.0 g，Na 2 HPO 4 0.2 g，MnSO 4 0.035 g，CuSO 4 ·5H 2 O 0.007 g，FeSO 4 ·7H 2 O 0.007 g。
Enzyme activity measurement: the strain MDD 2020 is inoculated into a triangular flask (250 mL) which is filled with 100 mL enzyme-producing medium, and is placed at 28 ℃ for 160 r min -1 Shaking culture 21d. Samples were taken 1 time per 2. 2 d, and the bacterial cells and impurities were removed by centrifugation to obtain a crude enzyme solution, and the activities of Lip (lignin peroxidase) and MnP (manganese peroxidase) in the crude enzyme solution were measured.
Solid state degradation assay: adding 5 g dry branch and fallen leaf powder (fallen leaves and broken branches of peach tree covered with upper layer of peach garden soil), distilled water washing, oven drying, pulverizing, sieving with 60 mesh sieve, and 10 mL solid culture nutrient solution, mixing the solid and liquid, making it flat, sterilizing, and inoculating 1% bacterial strain suspension of MDD 2020 (10) 7 Per mL) and then placed in an incubator at 30℃and 50℃for 30 d, respectively. During the incubation period, moisture was appropriately supplemented to maintain humidity. After the cultivation, the mixture was dried at 105℃until the weight was constant, and the weight loss rate of the dry-branch and fallen-leaf powder medium after 30 d was measured.
Example 1
The invention provides a bacterial strain capable of degrading agricultural dry branch and fallen leaf wastes, which belongs to the strain MDD 2020Ochrobactrumpseudintermeddium(pseudo-middle pallor) deposited in China general microbiological culture Collection center (CGMCC) with a culture collection number of CGMCC No.22199, 4.about.19.2021. The biological characteristics are as follows: on LB medium, at 37 ℃, 36h is cultivated, the colony is nearly round, milky white, moist on the surface, smooth and slightly convex, and has a single colony diameter of about 4mm, gram negative, positive for contact enzyme and oxidase. Indole was negative.
PCR amplification with bacterial Universal primers 27f and 1492r, sequence homology comparison in GenBank with BLAST, MDD 2020 andOchrobactrum pseudintermediumthe homology reaches 98%, a plurality of strains similar to the homology are selected, phylogenetic evolutionary trees are drawn, and the strains are identified as the strains by combining with morphological characteristics of bacterial coloniesOchrobactrum pseudintermeddium(Ochrobactrum pseudomiddle).
The strain MDD 2020 is enriched by a liquid enrichment medium and a solid enrichment medium which take the dry branch and fallen leaf powder as the only carbon source, and after enrichment, the strain is well grown and separated and purified on a guaiacol selection medium, the chromogenic reaction is detected on an LB-guaiacol plate, and the fading reaction is detected on an LB-aniline blue plate. Specific:
1) Enrichment of lignin high-efficiency degrading bacteria: and selecting a degrading bacterium sample, namely stacking dead branches and fallen leaves which are spoiled and crushed throughout the year, and collecting the degrading bacterium sample in a tin-free peach garden. The solid enrichment and the liquid enrichment which take the dry branch and fallen leaf powder as the only carbon source are respectively carried out for two continuous generations through 30 d. The strain with high-efficiency degradation effect on lignin in dead branches and fallen leaves is obtained.
The specific operation is as follows: mixing 0.2 g degrading bacteria sample in a solid medium of 20 g, adding 10 mL sterile water at a temperature of 30 ℃ for culturing 30 d at a constant temperature of 5 d, selecting a part with black degrading spots on the surface of the medium, re-inoculating the part into a new solid medium of 20 g, adding 10 mL sterile water at a temperature of 30 ℃ for continuously culturing 30 d at a constant temperature of 5 d, then selecting a liquid enrichment medium with 10 mL sterile water at a temperature of 30 ℃ for continuously culturing 30 d at a constant temperature of 5 d and 1 ml of liquid enrichment medium, re-inoculating the part with 10 mL sterile water at a temperature of 5 d into a new liquid enrichment medium of 100 mL, and continuously culturing 30 d at a temperature of 30 ℃ to form a suspension of enriched samples.
2) Separating and screening lignin degrading bacteria: diluting and separating the enriched sample suspension by using a gradient dilution coating method, wherein the specific operation is as follows: 1 mL to 9 mL sterile in water test tubes were aspirated from the enriched sample suspension, vortexed 10 s at a dilution gradient of 10 -1 1 mL was again taken into a new tube of 9 mL sterile water and vortexed 10 s at a dilution gradient of 10 -2 Sequentially diluting to 10 according to the method -4 .1 mug of diluted bacterial suspension is coated on guaiacol selection medium, and is cultivated at constant temperature of 30 ℃ for 2-3 d, strains growing faster are selected, repeated streaking separation is carried out until a pure strain is obtained, and the pure strain is preserved at 4 ℃ for standby.
The isolated strain was inoculated on LB-guaiacol plate and LB-aniline blue plate to observe the time and size of the development of the color circle and the color-fading circle, and the results are shown in FIG. 1. The strain MDD 2020 was found to have no chromogenic reaction on LB-guaiacol plates, but to produce a discoloured ring on LB-aniline blue plates. The phylogenetic tree results of this strain based on 16S rDNA sequence similarity are shown in FIG. 2.
3) Determination of degradation enzyme activity: bacterial strain suspension (10) of bacterial MDD 2020 was inoculated at 1% inoculum size 7 Each mL) was inoculated into a flask (250 mL) containing 100. 100 mL enzyme-producing medium, and the flask was subjected to shaking culture at 28℃for 160 r/min for 21d. Sampling every 2 times d, centrifuging the fermentation liquor, and removing thalli and impurities to obtain crude enzyme liquid. The activity of Lip (lignin peroxidase) and MnP (manganese peroxidase) was measured by inoculating into liquid enzyme to measure the change in the enzyme productivity of the strain in 21d. The enzyme activity of Lip is defined as oxidizing 1. Mu. Mol of veratrole per minute to one enzyme activity unit (U). The enzymatic activity of MnP is defined as oxidizing 1. Mu. MoL of Mn per minute 2+ Is Mn 3+ Is an enzyme activity unit (U).
And (3) measuring the activities of lignin peroxidase and manganese peroxidase in the crude enzyme liquid through liquid fermentation of 21d. The highest enzyme production peak of lignin peroxidase is at 12. 12 d, which is 18.46U mL -1 And then starts to descend. The highest peak of manganese peroxidase production is at 15 th d, which is 9.64U mL -1 The strain MDD 2020 can produce lignin degrading enzyme with strong capability, fast enzyme production and long enzyme production maintenance time.
4) Solid state degradation capability determination: adding 5 g dry branch and fallen leaf powder (fallen leaves and broken branches covered by the upper layer of peach garden soil), 10 mL solid culture nutrient solution, sterilizing, inoculating bacterial strain suspension (10) of bacterial MDD 2020 according to 1% inoculation amount 7 /mL) to solid degradation medium, and culturing the selected strain in an incubator at 30℃and 50℃for 30 d, respectively. During the incubation period, moisture was appropriately supplemented to maintain humidity. After the cultivation is finished, the mixture is dried to constant weight at 105 ℃. The degradation rate calculation method comprises the following steps:
degradation rate= (total mass before degradation-total mass after degradation)/5 g ×100%.
Solid-state degradation tests show that under the condition of 30 ℃, the solid-state degradation rate of 30 d can reach 8.67 percent when the culture medium is inoculated with the bacterium MDD 2020. At 50 ℃,30 d is degraded in solid state, and the degradation rate of the culture medium inoculated with the bacterium MDD 2020 can reach 8.33 percent.
The invention degrades agricultural dead branch and fallen leaf waste by utilizing a microbial strain MDD 2020 fermentation method, promotes the decomposition process of dead branch and fallen leaf compost, provides technical support for popularization and utilization of dead branch and fallen leaf compost, achieves the purposes of changing waste into valuables, protecting environment and promoting agricultural sustainable development, and has wide application prospect.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (2)
1. A bacterial strain capable of degrading agricultural dry branch and fallen leaf waste, which is characterized in that: the strain MDD 2020 belongs to the genus of the pseudo-middle pallor bacillus, is deposited in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has a culture Collection number of CGMCC No.22199.
2. Use of the bacterial strain of claim 1 for degrading agricultural deadwood defoliation waste.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106967637A (en) * | 2017-03-29 | 2017-07-21 | 南京农业大学 | One plant of pear tree branch bacterium for degrading L2 and its microbial inoculum |
| CN110669692A (en) * | 2019-10-22 | 2020-01-10 | 中国石油大学(华东) | Preparation method and application method of viscosity-reducing degradation mixed microbial inoculum for thickened oil |
| CN112522143A (en) * | 2020-12-07 | 2021-03-19 | 榆林鹤翼航天航空科技创新有限责任公司 | Novel kitchen waste high-temperature composting process assisted by complex enzyme preparation and ochrobactrum anthropi |
| CN113957002A (en) * | 2021-08-26 | 2022-01-21 | 中国林业科学研究院林产化学工业研究所 | Lignin degrading bacillus and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967637A (en) * | 2017-03-29 | 2017-07-21 | 南京农业大学 | One plant of pear tree branch bacterium for degrading L2 and its microbial inoculum |
| CN110669692A (en) * | 2019-10-22 | 2020-01-10 | 中国石油大学(华东) | Preparation method and application method of viscosity-reducing degradation mixed microbial inoculum for thickened oil |
| CN112522143A (en) * | 2020-12-07 | 2021-03-19 | 榆林鹤翼航天航空科技创新有限责任公司 | Novel kitchen waste high-temperature composting process assisted by complex enzyme preparation and ochrobactrum anthropi |
| CN113957002A (en) * | 2021-08-26 | 2022-01-21 | 中国林业科学研究院林产化学工业研究所 | Lignin degrading bacillus and application thereof |
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