Directional domestication method of acid-resistant cellulose degradation flora and application of acid-resistant cellulose degradation flora in phosphogypsum compost

Abstract

The invention discloses a directional domestication method of an acid-resistant cellulose degradation flora and application of the acid-resistant cellulose degradation flora in phosphogypsum composting, the directional domestication method comprises the steps of bacterium source collection, microorganism enrichment, acid screening and directional domestication, an acid screening culture medium is prepared from 1g / L of KH2PO4, 0.3 g / L of MgSO4. 7H2O, 0.1 g / L of CaCl2, 0.01 g / L of FeCl3, 2.5 g / L of NaNO3, 0.1 g / L of NaCl and 20-40g / L of waste biomass, and the pH is 3-6; a domestication culture medium is an acidic screening culture medium and 8-24g / L of phosphogypsum. The acclimatization is carried out to obtain the acid-resistant cellulose degradation flora, the degradation of waste biomass can be carried out in a composting system with the pH value of 3-6 and ardealite as a raw material, the acid-resistant cellulose degradation flora has the advantages of good acid tolerance, strong adaptability and low competitiveness, and the composting efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a directional domestication method of an acid-resistant cellulose-degrading bacterial community and an application thereof in phosphogypsum composting. Background Art

[0002] Phosphogypsum is an industrial waste byproduct of the decomposition of phosphate rock with sulfuric acid during the wet phosphoric acid production process. In addition to its main component, calcium sulfate dihydrate, phosphogypsum also contains phosphorus, sulfur, calcium, iron, zinc and other elements that promote plant growth. Therefore, phosphogypsum can be used to make soil conditioners and compound fertilizers and other related products. However, the composition of phosphogypsum is complex. The impurities such as phosphoric acid, phosphates, hydrogen fluoride, and heavy metals it contains make phosphogypsum subject to environmental risks such as heavy metal accumulation, excessive fluorine, and soil acidification in the process of agricultural resource utilization, which limits the application of phosphogypsum. At present, a large amount of phosphogypsum is mainly stored in piles, which not only occupies a large amount of land, but also brings huge pressure to the ecological environment around the storage yard. The resource recycling and reuse of phosphogypsum is the current key research direction.

[0003] Composting refers to the biochemical process of using widely existing microorganisms in nature to controllably promote the conversion of degradable organic matter in solid waste into stable humus. It has the characteristics of simple process, low cost, and environmental friendliness. The organic fertilizer formed is rich in nutrients and has a long and stable fertilizer effect. Based on the plant growth-promoting elements contained in phosphogypsum, composting with phosphogypsum as raw material shows great application potential.

[0004] Chinese invention patent CN104446716A discloses a method for preparing compost with phosphogypsum, which comprises mixing kitchen waste, corn stalks and phosphogypsum for composting, specifically utilizing SO4 2- PO4 3- The free phosphoric acid has a certain inhibitory and fixing effect on the production and emission of CH4 and NH3, which can significantly reduce the emission of CH4 and NH3 during the composting process. However, the fermentation time seems to be 28-35 days, which is a long period.

[0005] Microorganisms play an important role in the composting process. Increasing specific types of microorganisms can promote the decomposition of composting substrates and shorten the composting cycle. Chinese invention patent CN118324565A discloses a composting method for phosphogypsum, which inoculates microbial agents into phosphogypsum, adjusts the pH, adds promoters and microbial additives, and then mixes them with kitchen waste, feces, wood chips, straw, etc. for composting, wherein the microbial agents are selected from commercially available Pseudomonas alcaligenes, Citrobacter citriodora, Zymomonas, Acidophilus, and Bacillus coagulans.

[0006] Although there are many composting agents on the market, the composition of phosphogypsum is complex and highly acidic, and the inoculated agents are difficult to adapt to the acidic environment of the phosphogypsum composting system. Secondly, the inoculated agents will not only compete with the indigenous microorganisms in the composting materials, but also compete with each other. The inoculated agents need to be structurally adjusted at different stages of composting, resulting in low composting efficiency. Therefore, it is necessary to select appropriate inoculated agents based on the characteristics of phosphogypsum and composting materials to ensure composting efficiency. Summary of the invention

[0007] In view of the above technical problems, the present invention provides a method for the directed domestication of acid-resistant cellulose-degrading bacteria and its application in phosphogypsum composting. Acid-resistant cellulose-degrading bacteria are obtained by enriching and directed domestication in the humus layer soil of a phosphogypsum composting plant. The bacteria can be used in the composting of phosphogypsum to improve the efficiency of composting and the quality of composting.

[0008] In order to achieve the above object, the present invention provides a method for directional domestication of acid-resistant cellulose-degrading bacteria, comprising the following steps: (1) Collection of fungal sources: Collect humus soil from phosphogypsum pile plants; (2) Microbial enrichment: Mix the humus layer soil with sterile water to obtain a soil suspension. After standing, take the supernatant and mix it with LB medium to obtain a microbial enrichment culture solution. (3) Acid screening: Mix the microbial enrichment culture medium with the acid cellulose culture medium to obtain an acid-resistant cellulose-degrading bacterial flora enrichment culture medium; (4) Directed domestication: The enriched culture medium of the acid-resistant cellulose-degrading bacterial flora is mixed with the domestication medium, and the culture is carried out for 3-5 times to obtain domesticated acid-resistant cellulose-degrading bacterial flora.

[0009] Preferably, the phosphogypsum plant described in step (1) is located between N 30°15′29.13″-30°15′47.05″ and E 111°30′27.23″-111°31′05.18″.

[0010] Preferably, the acid cellulose culture medium in step (3) comprises 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 0.01 g / L FeCl3, 2.5 g / L NaNO3, 0.1 g / L NaCl, 20-40 g / L waste biomass, and a pH of 3-6.

[0011] Preferably, the acclimation medium formula of step (4) is 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 0.01 g / L FeCl3, NaNO32.5 g / L, NaCl 0.1 g / L, waste biomass 20-40 g / L, phosphogypsum 8-24 g / L, and pH 3-6.

[0012] Further preferably, the waste biomass is any one or more of straw, bran and vegetable leaves.

[0013] Further preferably, the particle size of the phosphogypsum is 1-5 mm, and the particle size of the waste biomass is 2-5 cm.

[0014] Preferably, the content of phosphogypsum in the acclimation culture medium used in each acclimation culture in step (4) is increased by 1-4 g / L compared with the amount of phosphogypsum in the acclimation culture medium used in the previous acclimation culture; and the pH is reduced by 0-2.

[0015] Preferably, the mass ratio of the humus soil to sterile water in step (2) is 0.5-1:1; the volume ratio of the supernatant to the LB medium is 0.2-0.5:1; the volume ratio of the microbial enrichment culture solution to the acid cellulose medium in step (3) is 0.2-0.5:1; the volume ratio of the acid-resistant cellulose degrading bacterial enrichment culture solution to the acclimation medium in step (4) is 0.2-0.5:1.

[0016] Preferably, the domesticated acid-resistant cellulose-degrading bacterial flora is any one or more of the genera Sphingomonas, Streptomyces, Ramlibacter, Trebonia, Marmoricola, Nocardioides, Escherichia, Pseudolabrys, Bradyrhizobium and Occallatibacter.

[0017] Further preferably, when the acid cellulose medium is prepared with straw as the carbon source, the domesticated acid-resistant cellulose-degrading bacteria mainly include Sphingomonas, Ramlibacter, Trebonia, Nocardioides, Escherichia, Pseudolabrys, Bradyrhizobium and Occallatibacter; When the acid cellulose medium was prepared with straw and vegetable leaves as carbon sources, the domesticated acid-resistant cellulose-degrading bacteria mainly included Sphingomonas, Streptomyces, Ramlibacter, Marmoricola, Nocardioides, Pseudolabrys, Bradyrhizobium and Occallatibacter. When the acid cellulose culture medium was prepared with a combination of straw, vegetable leaves and bran as carbon sources, the domesticated acid-resistant cellulose-degrading bacteria mainly included Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides and Bradyrhizobium.

[0018] Further preferably, when the combination of straw and vegetable leaves is used as the carbon source, the mass ratio of straw to vegetable leaves is 1-5:1-5; when the combination of straw, vegetable leaves and bran is used as the carbon source, the mass ratio of straw, vegetable leaves and bran is 1-5:1-5:1-5.

[0019] Preferably, the culture conditions of the soil suspension in step (2) are 28-32°C, 120-150r / min shaking culture for 15-30min; the culture conditions of the microbial enrichment culture solution are 28-32°C, 120-150r / min shaking culture for 5-7d.

[0020] Preferably, the standing time in step (2) is 1-2 hours.

[0021] Preferably, the culture conditions of the acid screening in step (3) and the acclimation culture in step (4) are both 28-32° C. and 120-150 r / min shaking culture for 5-7 days.

[0022] The present invention also provides an application of an acid-resistant cellulose-degrading bacterial community in phosphogypsum composting, wherein the acid-resistant cellulose-degrading bacterial community is domesticated according to the above-mentioned directional domestication method; the method for composting the phosphogypsum is: mixing phosphogypsum with waste biomass to obtain a compost material, adding an acid-resistant cellulose-degrading bacterial community liquid to the compost material, and performing composting fermentation.

[0023] Preferably, the mass ratio of the phosphogypsum to the waste biomass is 0.4-0.6:1, the mass ratio of the acid-resistant cellulose-degrading bacterial liquid to the compost material is 0.01-0.05:1; the concentration of the acid-resistant cellulose-degrading bacterial liquid is 1×10 6 -1×10 8 CFU / mL.

[0024] Preferably, before composting, the pH of the composting system is 3.0-6.0, the carbon-nitrogen ratio is 25-35:1, the carbon-phosphorus ratio is 75-150:1, the humidity is 50-60%, and the oxygen content is 10-18%.

[0025] Preferably, the particle size of the phosphogypsum is 50-100 mm; and the waste biomass is any one or more of straw, bran and vegetable leaves.

[0026] The beneficial effects of the present invention are: 1. Using the humus soil of the phosphogypsum plant as the bacterial source, an acid-resistant cellulose-degrading bacterial community was obtained through enrichment, acid screening and directional domestication and separation. The community can degrade waste biomass in a composting system with a pH of 3-6 and phosphogypsum as raw material. The community has good acid tolerance, strong adaptability, and will not compete with indigenous microorganisms in the compost material. There will also be no competition between the acid-resistant cellulose-degrading bacterial communities after inoculation, thereby improving the application efficiency.

[0027] 2. The present invention selectively screens highly efficient indigenous acid-resistant cellulose-degrading bacteria according to the types of waste biomass in the composting system, thereby improving the composting efficiency and effect, and making the content of dissolved organic matter in the composting system reach 650-830 g / kg, which has more advantages in terms of cost, efficiency, equipment investment, etc.

[0028] 3. The present invention utilizes indigenous acid-resistant cellulose-degrading bacteria from humus soil in phosphogypsum storage yards to decompose waste biomass in the composting system, giving full play to the multiple advantages of microorganisms such as low cost, high efficiency and good effect, and provides a practical solution to solve the huge problem of phosphogypsum storage in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing the genus distribution of the acid-resistant cellulose-degrading bacterial community domesticated in Example 1.

[0030] Figure 2 This is a diagram showing the genus distribution of the acid-resistant cellulose-degrading bacterial community domesticated in Example 3.

[0031] Figure 3 This is a diagram showing the genus distribution of the acid-resistant cellulose-degrading bacterial community domesticated in Example 5.

[0032] Figure 4 This is a diagram showing the genus distribution of the acid-resistant cellulose-degrading bacterial community domesticated in Example 6. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

[0034] In the following examples, the formula of LB medium is peptone 10.0 g / L, NaCl 10.0 g / L, yeast extract powder 5.0 g / L, pH 6.5-7.5, the solvent is deionized water, and the pH is natural; Phosphogypsum is a byproduct of the calcium sulfate dihydrate process in the phosphoric acid workshop of Yidu Xingfa Chemical Co., Ltd. in Yichang City, Hubei Province. It contains 68.3% calcium sulfate dihydrate, 25.6% attached water, 1.0% water-soluble phosphorus, and 0.9% water-soluble fluorine. When preparing the culture medium, the particle size of phosphogypsum is 3 mm and the particle size of the waste biomass is 2 cm; when composting, the particle size of phosphogypsum is 80 mm and the particle size of the waste biomass is 3 cm.

[0035] Example 1 (1) Bacteria source collection: The humus layer soil of the phosphogypsum plant (N 30°15′29.13″, E 111°30′27.23″) was taken as the bacteria source sample; (2) Obtaining soil suspension: Mix humus soil and sterile water at a mass ratio of 0.5:1, shake at 28°C and 120 rpm for 15 min, and let stand for 1 h to obtain soil suspension; (3) Microbial enrichment: Take the supernatant of the soil suspension and mix it with LB medium at a volume ratio of 0.2:1, shake it at 25°C and 120 rpm for 5 days to obtain a microbial enrichment culture solution; (4) Preparation of culture medium: Acid cellulose medium: formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.3g / L CaCl2·2H2O, 0.01g / L FeCl3, NaNO32.5g / L, NaCl 0.1g / L, 20g / L straw, pH is 6; Acclimation medium: formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / LFeCl3, NaNO32.5g / L, NaCl 0.1g / L, 20g / L straw, 8-12g / L phosphogypsum, pH 3-6; (5) Acid screening: The microbial enrichment culture medium was mixed with the acid cellulose culture medium at a volume ratio of 0.2:1, and shaken at 25°C and 120 r / min for 5 days to obtain an acid-resistant cellulose-degrading bacterial enrichment culture medium; (6) Directed domestication: Primary acclimation: The enriched culture solution of acid-resistant cellulose-degrading bacteria was mixed with the acclimation medium at a volume ratio of 0.2:1, and the mixture was shaken at 28°C and 120 r / min for 7 days to obtain the primary acclimation culture solution, wherein the amount of phosphogypsum in the acclimation medium was 8 g / L and the pH was 6; Secondary domestication: The method is the same as the primary domestication, with the domestication time of 6 days, the dosage of phosphogypsum in the domestication medium of 9 g / L, and the pH of 5; Three domestication: the method is the same as the first domestication, wherein the amount of phosphogypsum in the domestication medium is 12 g / L, the pH is 3, and the domesticated acid-resistant cellulose-degrading bacterial flora is obtained; (7) Humus soil from five different locations in the plant was collected for bacterial strain screening and domestication. After domestication, five parallel samples of bacterial liquid from each location were collected for metagenomic sequencing to identify key species. The frequency of species occurrence in each parallel sample was counted and summarized as the key species at that location. Then, the main bacterial species screened and domesticated at the five different locations were counted.

[0036] The main bacterial species in the acid-resistant cellulose-degrading bacterial community were identified by metagenomic sequencing. Figure 1 As shown, the main bacterial species include Sphingomonas, Streptomyces, Ramlibacter, Marmoricola, Nocardioides, Pseudolabrys, Bradyrhizobium and Occallatibacter Example 2 (1) The acid-resistant cellulose-degrading bacterial flora obtained in Example 1 was cultured to obtain an acid-resistant cellulose-degrading bacterial flora solution, and the concentration was adjusted to 1×10 6 CFU / mL; (2) mixing phosphogypsum and straw in a mass ratio of 0.4:1 to obtain compost material, wherein the carbon-nitrogen ratio is 25:1 and the carbon-phosphorus ratio is 75:1; (3) adding acid-resistant cellulose-degrading bacterial flora to the compost material, adjusting the system pH to 6, humidity to 50%, and oxygen content to 10% for composting, wherein the mass ratio of the acid-resistant cellulose-degrading bacterial flora to the compost material is 0.01:1; (4) After 15 days of composting, the compost material was air-dried naturally, and 10 g of the sample was weighed into a 100 mL centrifuge tube after being sieved through a 2 mm sieve. 50 mL of deionized water was added at a water-to-soil ratio of 5:1. After being fully shaken, the compost was shaken at 30°C, 200 r / min, and protected from light for 24 h. Then, the compost was centrifuged at 4,000 r / min for 15 min. After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane, and the dissolved organic matter content was determined using a UV spectrophotometer. The results showed that the dissolved organic matter content reached 668 g / kg.

[0037] Example 3 (1) Bacteria source collection: Soil from the humus layer of the phosphogypsum plant (N 30°15′30.00″, E 111°30′28.26″) was used as the bacteria source sample; (2) Obtaining soil suspension: Mix humus soil and sterile water at a mass ratio of 0.75:1, shake at 30°C and 130 rpm for 20 min, and let stand for 1 h to obtain soil suspension; (3) Microbial enrichment: Take the supernatant of the soil suspension and mix it with LB medium at a volume ratio of 0.3:1, shake it at 28°C and 130 rpm for 5 days to obtain a microbial enrichment culture solution; (4) Preparation of culture medium: Acid cellulose medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / L FeCl3, NaNO32.5g / L, NaCl 0.1g / L, 30g / L waste biomass, pH is 5; the waste biomass is composed of straw and vegetable leaves in a mass ratio of 1:1; Acclimation medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / LFeCl3, NaNO32.5g / L, NaCl 0.1g / L, 30g / L waste biomass, 12-18g / L phosphogypsum, pH 3-5; wherein the waste biomass is composed of straw and vegetable leaves in a mass ratio of 1:1; (5) Acid screening: The microbial enrichment culture medium was mixed with the acid cellulose culture medium at a volume ratio of 0.3:1, and shaken at 30°C and 130 r / min for 5 days to obtain an acid-resistant cellulose-degrading bacterial flora enrichment culture medium; (6) Directed domestication: Primary acclimation: The enriched culture solution of acid-resistant cellulose-degrading bacteria was mixed with the acclimation medium at a volume ratio of 0.25:1, and the mixture was shaken at 30°C and 120 r / min for 7 days to obtain the primary acclimation culture solution, wherein the amount of phosphogypsum in the acclimation medium was 12 g / L and the pH was 5; Secondary domestication: The method is the same as the primary domestication, with the domestication time of 6 days, the dosage of phosphogypsum in the domestication medium of 14 g / L, and the pH of 5; Three domestication: the method is the same as the first domestication, wherein the amount of phosphogypsum in the domestication medium is 18 g / L, the pH is 3, and the domesticated acid-resistant cellulose-degrading bacterial flora is obtained; (7) Humus soil from five different locations in the plant was collected for bacterial strain screening and domestication. After domestication, five parallel samples of bacterial liquid from each location were collected for metagenomic sequencing to identify key species. The frequency of species occurrence in each parallel sample was counted and summarized as the key species at that location. Then, the main bacterial species screened and domesticated at the five different locations were counted.

[0038] The main bacterial species in the acid-resistant cellulose-degrading bacterial community were identified by metagenomic sequencing. Figure 2 As shown, the main bacterial species include Sphingomonas, Streptomyces, Ramlibacter, Marmoricola, Nocardioides, Pseudolabrys, Bradyrhizobium and Occallatibacter; Example 4 (1) The acid-resistant cellulose-degrading bacterial flora obtained in Example 3 was cultured to obtain an acid-resistant cellulose-degrading bacterial flora solution, and the concentration was adjusted to 1×10 6 CFU / mL; (2) mixing phosphogypsum and waste biomass at a mass ratio of 0.4:1 to obtain compost material, wherein the carbon-nitrogen ratio is 25:1 and the carbon-phosphorus ratio is 75:1; wherein the waste biomass is composed of straw and vegetable leaves at a mass ratio of 1:1; (3) adding acid-resistant cellulose-degrading bacterial flora to the compost material, adjusting the system pH to 6, humidity to 50%, and oxygen content to 10% for composting, wherein the mass ratio of the acid-resistant cellulose-degrading bacterial flora to the compost material is 0.01:1; (4) After 15 days of composting, the compost material was air-dried naturally, and 10 g of the sample was weighed into a 100 mL centrifuge tube after being sieved through a 2 mm sieve. 50 mL of deionized water was added at a water-to-soil ratio of 5:1. After being fully shaken, the compost was shaken at 30°C, 200 r / min, and protected from light for 24 h. Then, the compost was centrifuged at 4,000 r / min for 15 min. After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane, and the dissolved organic matter content was determined using a UV spectrophotometer. The results showed that the dissolved organic matter content reached 723 g / kg.

[0039] Example 5 (1) Bacteria source collection: Soil from the humus layer of the phosphogypsum plant (N 30°15′30.00″, E 111°30′29.21″) was used as the bacteria source sample; (2) Obtaining soil suspension: Mix humus soil and sterile water at a mass ratio of 0.8:1, shake at 30°C and 140 rpm for 25 min, and let stand for 1.5 h to obtain soil suspension; (3) Microbial enrichment: Take the supernatant of the soil suspension and mix it with LB medium at a volume ratio of 0.4:1, shake it at 30°C and 140 rpm for 5 days to obtain a microbial enrichment culture solution; (4) Preparation of culture medium: Acid cellulose medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / L FeCl3, NaNO32.5g / L, NaCl 0.1g / L, 40g / L waste biomass, pH is 5; the waste biomass is composed of straw, vegetable leaves, and bran in a mass ratio of 1:1:1; Acclimation medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / LFeCl3, NaNO32.5g / L, NaCl 0.1g / L, 30g / L waste biomass, 16-24g / L phosphogypsum, pH 3-5; wherein the waste biomass is composed of straw, vegetable leaves, and bran in a mass ratio of 1:1:1; (5) Acid screening: The microbial enrichment culture medium was mixed with the acid cellulose culture medium at a volume ratio of 0.4:1, and shaken at 30°C and 140 rpm for 5 days to obtain an acid-resistant cellulose-degrading bacterial enrichment culture medium; (6) Directed domestication: Primary acclimation: The enriched culture solution of acid-resistant cellulose-degrading bacteria was mixed with the acclimation medium at a volume ratio of 0.4:1, and the mixture was shaken at 30°C and 140 r / min for 5 days to obtain the primary acclimation culture solution, wherein the amount of phosphogypsum in the acclimation medium was 16 g / L and the pH was 5; Secondary domestication: The method is the same as the primary domestication, with the domestication time of 6 days, the dosage of phosphogypsum in the domestication medium is 20 g / L, and the pH is 4.5; Three domestication: the method is the same as the first domestication, wherein the amount of phosphogypsum in the domestication medium is 24 g / L, the pH is 3, and the domesticated acid-resistant cellulose-degrading bacterial flora is obtained; (7) Humus soil from five different locations in the plant was collected for bacterial strain screening and domestication. After domestication, five parallel samples of bacterial liquid from each location were collected for metagenomic sequencing to identify key species. The frequency of species occurrence in each parallel sample was counted and summarized as the key species at that location. Then, the main bacterial species screened and domesticated at the five different locations were counted.

[0040] The main bacterial species in the acid-resistant cellulose-degrading bacterial community were identified by metagenomic sequencing. Figure 3 As shown, the bacterial species mainly include Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides and Bradyrhizobium.

[0041] Example 6 (1) The acid-resistant cellulose-degrading bacterial flora obtained in Example 5 was cultured to obtain an acid-resistant cellulose-degrading bacterial flora solution, and the concentration was adjusted to 1×10 7 CFU / mL; (2) mixing phosphogypsum and waste biomass at a mass ratio of 0.4:1 to obtain compost material, wherein the carbon-nitrogen ratio is 25:1 and the carbon-phosphorus ratio is 75:1; wherein the waste biomass is composed of straw, vegetable leaves and bran at a mass ratio of 1:1:1; (3) adding acid-resistant cellulose-degrading bacterial flora to the compost material, adjusting the system pH to 6, humidity to 50%, and oxygen content to 10% for composting, wherein the mass ratio of the acid-resistant cellulose-degrading bacterial flora to the compost material is 0.01:1; (4) After 15 days of composting, the compost was naturally air-dried, and 10 g of the sample was weighed into a 100 mL centrifuge tube after being sieved through a 2 mm sieve. 50 mL of deionized water was added at a water-to-soil ratio of 5:1. After being fully shaken, the compost was shaken at 30°C, 200 r / min, and protected from light for 24 h. The supernatant was then centrifuged at 4,000 r / min for 15 min. After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane, and the dissolved organic matter content was determined using a UV spectrophotometer. The results showed that the dissolved organic matter content reached 763 g / kg.

[0042] Example 7 (1) Bacteria source collection: Soil from the humus layer of the phosphogypsum plant (N 30°15′32.01″, E 111°30′29.56″) was used as the bacteria source sample; (2) Obtaining soil suspension: Mix humus soil and sterile water in a mass ratio of 1:1, shake at 32°C and 150 rpm for 30 min, and let stand for 1 h to obtain soil suspension; (3) Microbial enrichment: Take the supernatant of the soil suspension and mix it with LB medium at a volume ratio of 0.5:1, shake it at 32°C and 150 rpm for 7 days to obtain a microbial enrichment culture solution; (4) Preparation of culture medium: Acid cellulose medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / L FeCl3, NaNO32.5g / L, NaCl 0.1g / L, 40g / L waste biomass, pH is 4.5; the waste biomass is composed of straw, vegetable leaves, and bran in a mass ratio of 2:1:1; Acclimation medium: the formula is 1g / L KH2PO4, 0.3g / L MgSO4·7H2O, 0.1g / L CaCl2, 0.01g / LFeCl3, NaNO32.5g / L, NaCl 0.1g / L, 30g / L waste biomass, 16-24g / L phosphogypsum, pH 3-5; wherein the waste biomass is composed of straw, vegetable leaves, and bran in a mass ratio of 2:1:1; (5) Acid screening: The microbial enrichment culture medium was mixed with the acid cellulose culture medium at a volume ratio of 0.5:1, and shaken at 32°C and 150 rpm for 5 days to obtain an acid-resistant cellulose-degrading bacterial enrichment culture medium; (6) Directed domestication: Primary acclimation: The enriched culture solution of acid-resistant cellulose-degrading bacteria was mixed with the acclimation medium at a volume ratio of 0.5:1, and the mixture was shaken at 30°C and 140 r / min for 5 days to obtain the primary acclimation culture solution, wherein the amount of phosphogypsum in the acclimation medium was 16 g / L and the pH was 4.5; Secondary domestication: The method is the same as the primary domestication, wherein the speed during domestication is 120r / min, the amount of phosphogypsum in the domestication medium is 22g / L, and the pH is 4.5; Three domestication: the method is the same as the first domestication, wherein the amount of phosphogypsum in the domestication medium is 24 g / L, the pH is 3, and the domesticated acid-resistant cellulose-degrading bacterial flora is obtained; (7) Humus soil from five different locations in the plant was collected for bacterial strain screening and domestication. After domestication, five parallel samples of bacterial liquid from each location were collected for metagenomic sequencing to identify key species. The frequency of species occurrence in each parallel sample was counted and summarized as the key species at that location. Then, the main bacterial species screened and domesticated at the five different locations were counted.

[0043] The main bacterial species in the acid-resistant cellulose-degrading bacterial community were identified by metagenomic sequencing. Figure 4As shown, the bacterial species mainly include Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, Solirubrobacter, and Rhizobium.

[0044] Example 8 (1) The acid-resistant cellulose-degrading bacterial flora obtained in Example 7 was cultured to obtain an acid-resistant cellulose-degrading bacterial flora solution, and the concentration was adjusted to 1×10 8 CFU / mL; (2) mixing phosphogypsum and waste biomass at a mass ratio of 0.4:1 to obtain compost material, wherein the carbon-nitrogen ratio is 25:1 and the carbon-phosphorus ratio is 75:1; wherein the waste biomass is composed of straw, vegetable leaves and bran at a mass ratio of 2:1:1; (3) adding acid-resistant cellulose-degrading bacterial flora to the compost material, adjusting the system pH to 6, humidity to 50%, and oxygen content to 10% for composting, wherein the mass ratio of the acid-resistant cellulose-degrading bacterial flora to the compost material is 0.01:1; (4) After 15 days of composting, the compost material was air-dried naturally, and 10 g of the sample was weighed into a 100 mL centrifuge tube after being sieved through a 2 mm sieve. 50 mL of deionized water was added at a water-to-soil ratio of 5:1. After being fully shaken, the compost was shaken at 30 °C, 200 r / min, and protected from light for 24 h. Then, the compost was centrifuged at 4,000 r / min for 15 min. After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane, and the dissolved organic matter content was determined using a UV spectrophotometer. The results showed that the dissolved organic matter content reached 827 g / kg.

[0045] Example 9 (1) The acid-resistant cellulose-degrading bacterial flora obtained in Example 1 was cultured to obtain an acid-resistant cellulose-degrading bacterial flora solution, and the concentration was adjusted to 1×10 8 CFU / mL; (2) mixing phosphogypsum and straw in a mass ratio of 0.5:1 to obtain compost material, wherein the carbon-nitrogen ratio is 28:1 and the carbon-phosphorus ratio is 90:1; (3) adding acid-resistant cellulose-degrading bacterial flora to the compost material, adjusting the system pH to 5, humidity to 55%, and oxygen content to 15% for composting, wherein the mass ratio of acid-resistant cellulose-degrading bacterial flora to compost material is 0.03:1; (4) After 15 days of composting, the compost material was air-dried naturally, and 10 g of the sample was weighed into a 100 mL centrifuge tube after being sieved through a 2 mm sieve. 50 mL of deionized water was added at a water-to-soil ratio of 5:1. After being fully shaken, the compost was shaken at 30°C, 200 r / min, and protected from light for 24 h. Then, the compost was centrifuged at 4,000 r / min for 15 min. After centrifugation, the supernatant was filtered through a 0.45 μm filter membrane, and the dissolved organic matter content was determined using a UV spectrophotometer. The results showed that the dissolved organic matter content reached 864 g / kg.

[0046] Example 10 The method and steps are the same as those of Example 9, except that the mass ratio of phosphogypsum to straw in step (2) is changed to 0.6:1, the carbon-nitrogen ratio is changed to 30:1, and the phosphorus-carbon ratio is changed to 100:1; In step (3), the mass ratio of the acid-resistant cellulose-degrading bacterial culture to the compost material is 0.04:1.

[0047] The results showed that the content of dissolved organic matter reached 852g / kg.

[0048] Embodiment 11 The method and steps are the same as those of Example 9, except that the mass ratio of phosphogypsum to straw in step (2) is changed to 0.6:1, the carbon-nitrogen ratio is changed to 35:1, and the phosphorus-carbon ratio is changed to 120:1; In step (3), the system pH is 3, the humidity is 60%, the oxygen content is 20%, and the mass ratio of the acid-resistant cellulose-degrading bacterial liquid to the compost material is 0.05:1.

[0049] The results showed that the content of dissolved organic matter reached 889g / kg.

[0050] Comparative Example 1 (1) The method and steps are the same as those of Example 7, except that the amount of waste biomass in the acidic cellulose medium in step (4) is changed to 15 g / L, and the main bacterial species screened and domesticated are Sphingomonas, Occallatibaca, Streptomyces, Escherichia, Ramlibacter, Bradyrhizobium, Solirubrobacter, and Hyphomicobium; (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0051] The results showed that the content of dissolved organic matter was 543 g / kg.

[0052] Comparative Example 2 (1) The method and steps are the same as those of Example 7, except that the amount of waste biomass in the acidic cellulose medium in step (4) is changed to 45 g / L, and the main bacterial species screened and domesticated are Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, and solinubrobacter; (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0053] The results showed that the content of dissolved organic matter was 682g / kg.

[0054] Comparative Example 3 (1) The method and steps are the same as those in Example 7, except that the directional domestication in step (6) is changed to a single domestication, wherein the amount of phosphogypsum in the domestication medium is 16 g / L, the pH is 4.5, and the main bacterial species screened and domesticated are Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, Solirubrobacter, Rhizobium, Mesorhizobium, and Actinoplanes; (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0055] The results showed that the content of dissolved organic matter was 521 g / kg.

[0056] Comparative Example 4 (1) The method and steps are the same as those in Example 7, except that the directional acclimation in step (6) is changed to two acclimations, wherein one acclimation is as follows: the enriched culture solution of the acid-resistant cellulose-degrading bacterial community is mixed with the acclimation medium at a volume ratio of 0.5:1, and the mixture is shaken at 30° C. and 140 r / min for 5 days to obtain a primary acclimation culture solution, wherein the amount of phosphogypsum in the acclimation medium is 16 g / L, and the pH is 4.5; Secondary domestication: The method was the same as the primary domestication, except that the speed was 120 r / min, the amount of phosphogypsum in the domestication medium was 22 g / L, the pH was 4.5, and the main strains screened and domesticated were Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, Lutetalea, and Trebonia. (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0057] The results showed that the content of dissolved organic matter was 692g / kg.

[0058] Comparative Example 5 (1) The method and steps are the same as those in Example 7, except that in step (6), the amount of phosphogypsum in the first domestication is changed to 8 g / L, and the pH is changed to 6; the amount of phosphogypsum in the second domestication is changed to 13 g / L, and the pH is changed to 3; the amount of phosphogypsum in the third domestication is changed to 18 g / L, and the pH is changed to 3; the main strains obtained by screening and domestication are Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, and Mesorhizobium; (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0059] The results showed that the content of dissolved organic matter was 686 g / kg.

[0060] Comparative Example 6 (1) The method and steps are the same as those in Example 7, except that the amount of phosphogypsum used in the first domestication in step (6) is changed to 8 g / L, the amount of phosphogypsum used in the second domestication is changed to 13 g / L, and the amount of phosphogypsum used in the third domestication is changed to 18 g / L. The main strains obtained by screening and domestication are Sphingomonas, Streptomyces, Escherichia, Nocardioides, Bradyrhizobium, Solirubrobacter, and Pseudolabrys. (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0061] The results showed that the content of dissolved organic matter was 663g / kg.

[0062] Comparative Example 7 (1) The method and steps are the same as those in Example 7, except that the pH value in the first acclimation is changed to 6, the pH value in the second acclimation is changed to 3, and the pH value in the third acclimation is changed to 3 in step (6); the main bacterial species obtained by screening and acclimation are Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides, Bradyrhizobium, Hyphomiarobium, and Phenylobacterium; (2) Cultivate the main bacterial strains to obtain bacterial liquid, and compost it according to the method of Example 8.

[0063] The results showed that the content of dissolved organic matter was 708g / kg.

[0064] Comparative Example 8 The method and steps are the same as steps (1) to (3) in Example 7 to obtain a microbial enrichment culture solution, and after adjusting the concentration, composting is directly carried out according to the method of Example 8.

[0065] The results showed that the content of dissolved organic matter was 244 g / kg.

Claims

1. A method for directional domestication of acid-resistant cellulose-degrading bacteria, characterized in that: The steps include: (1) Collection of fungal sources: Collect humus soil from phosphogypsum pile plants; (2) Microbial enrichment: Mix the humus layer soil with sterile water to obtain a soil suspension. After standing, take the supernatant and mix it with LB medium to obtain a microbial enrichment culture solution. (3) Acid screening: Mix the microbial enrichment culture medium with the acid cellulose culture medium to obtain an acid-resistant cellulose-degrading bacterial flora enrichment culture medium; (4) Directed domestication: The enriched culture medium of the acid-resistant cellulose-degrading bacterial flora is mixed and cultured with the domestication medium, and the domestication culture is repeated 3 to 5 times to obtain domesticated acid-resistant cellulose-degrading bacterial flora.

2. The method for directed domestication of an acid-resistant cellulose-degrading bacterial community according to claim 1, characterized in that: The formula of the acid cellulose culture medium in step (3) is 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 0.01 g / L FeCl3, 2.5 g / L NaNO3, 0.1 g / L NaCl, 20-40 g / L waste biomass, and pH 3-6.

3. The method for directed domestication of an acid-resistant cellulose-degrading bacterial community according to claim 1, characterized in that: The acclimation medium formula of step (4) is acidic screening medium and 8-24 g / L phosphogypsum, with a pH of 3-6.

4. The method for directional domestication of an acid-resistant cellulose-degrading bacterial community according to claim 2 or 3, characterized in that: The waste biomass is any one or more of straw, bran and vegetable leaves.

5. The method for directed domestication of acid-resistant cellulose-degrading bacteria according to claim 1, characterized in that: The content of phosphogypsum in the acclimation culture medium used in each acclimation culture in step (4) is increased by 1-4 g / L compared with the amount of phosphogypsum in the acclimation culture medium used in the previous acclimation culture; and the pH is reduced by 0-2.

6. The method for directed domestication of acid-resistant cellulose-degrading bacteria according to claim 1, characterized in that: The volume ratio of the microbial enrichment culture solution to the acid cellulose culture medium in step (3) is 0.2-0.5:1; the volume ratio of the acid-resistant cellulose degrading bacterial community enrichment culture solution to the acclimation culture medium in step (4) is 0.2-0.5:

1.

7. The directed domestication method according to any one of claims 1 to 6, characterized in that: The domesticated acid-resistant cellulose-degrading bacterial flora is any one or more of the genera Sphingomonas, Streptomyces, Ramlibacter, Trebonia, Marmoricola, Nocardioides, Escherichia, Pseudolabrys, Bradyrhizobium and Occallatibacter.

8. The directed domestication method according to claim 7, characterized in that: When straw was used as the carbon source to prepare the acid cellulose medium, the domesticated acid-tolerant cellulose-degrading bacteria mainly included Sphingomonas, Ramlibacter, Trebonia, Nocardioides, Escherichia, Pseudolabrys, Bradyrhizobium and Occallatibacter. When the acid cellulose medium was prepared with straw and vegetable leaves as carbon sources, the domesticated acid-resistant cellulose-degrading bacteria mainly included Sphingomonas, Streptomyces, Ramlibacter, Marmoricola, Nocardioides, Pseudolabrys, Bradyrhizobium and Occallatibacter. When the acid cellulose culture medium was prepared with a combination of straw, vegetable leaves and bran as carbon sources, the domesticated acid-resistant cellulose-degrading bacteria mainly included Sphingomonas, Streptomyces, Escherichia, Ramlibacter, Nocardioides and Bradyrhizobium.

9. An application of an acid-resistant cellulose-degrading bacterial community in phosphogypsum composting, characterized in that: The acid-resistant cellulose-degrading bacterial community is domesticated according to the directional domestication method described in any one of claims 1 to 6; the method for composting phosphogypsum is: mixing phosphogypsum with waste biomass to obtain compost material, adding acid-resistant cellulose-degrading bacterial community liquid to the compost material, and composting and fermenting.

10. The use according to claim 9, characterized in that: The mass ratio of the phosphogypsum to the waste biomass is 0.4-0.6:1, the mass ratio of the acid-resistant cellulose-degrading bacterial liquid to the compost material is 0.01-0.05:1; the concentration of the acid-resistant cellulose-degrading bacterial liquid is 1×10 6 -1×10 8 CFU / mL.

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