CN116622519A - Composite microbial agent and preparation method and application thereof - Google Patents
Composite microbial agent and preparation method and application thereof Download PDFInfo
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- CN116622519A CN116622519A CN202310491419.0A CN202310491419A CN116622519A CN 116622519 A CN116622519 A CN 116622519A CN 202310491419 A CN202310491419 A CN 202310491419A CN 116622519 A CN116622519 A CN 116622519A
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- 238000002360 preparation method Methods 0.000 title claims description 8
- 239000010815 organic waste Substances 0.000 claims abstract description 33
- 241000179039 Paenibacillus Species 0.000 claims abstract description 24
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 22
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- 238000000034 method Methods 0.000 claims abstract description 20
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention provides a composite microbial agent and application thereof, and belongs to the technical field of microorganisms. The composite microbial agent of the invention comprises aspergillus niger, pseudo-pallidum, paenibacillus xylan, bacillus subtilis and paenibacillus amyloliquefaciensThe total viable bacteria concentration of the composite microbial agent is 2 multiplied by 10 8 ～2×10 10 cfu/ml. The composite microbial agent provided by the invention has stronger degradation capability on lignocellulose and no antagonism among strains, and can accelerate the composting process of agricultural organic wastes, promote composting maturity and improve the quality of composting products.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a composite microbial agent, a preparation method and application thereof.
Background
The increase of global population and the improvement of living conditions lead to the continuous increase of the demand for grain production, bring great pressure to agricultural production, and statistically generate 1400 hundred million tons of lignocellulose related agricultural organic waste every year in the global scope, and the agricultural waste generated in China accounts for 1/10 of the total amount of the agricultural waste. As a reusable resource, the inability to properly dispose of a large number of agricultural byproducts not only results in waste of resources, but also can cause pollution to surface and ground water, and the generation of a large amount of greenhouse gases, resulting in pollution of air, so how to properly and safely dispose of agricultural organic waste is a global challenge.
In order to realize the recycling of resources, the agricultural organic waste is inoculated with microbial agents and then is subjected to composting treatment to become the organic fertilizer rich in nutrient substances. The microbial inoculant inoculation treatment of agricultural organic waste can be traced to the 90 s of the 19 th century, and the Yang et al inoculated Trametes hirsuta S and Pleurotus ostreatus S to tobacco straw to increase lignin degradation from 23.7% to 41.1%. Chu et al constructed composite strains as Phanerochaete chrysosporium, trametes ersicolor and Pleurotus ostreatus with degradation rates of 43.36%, 31.29% and 48.36% for lignin, cellulose and hemicellulose, respectively. After fermentation of Pseudomonas (Pseudoxanthomonas sp) R-28 screened by Kumar et al for 5d, the degradation efficiency on filter paper and pure cellulose waste materials is 96% and 95%, respectively; the new tolumonas (Toluomonas) strain which takes lignin as a main carbon source is obtained by separating the seeds of the tilling and the like from tropical rainforest soil, and has stronger lignin peroxidase activity. Wan et al have discovered that the addition of microbial agents during composting can significantly accelerate the degradation rate of organic waste.
The firm nature of lignocellulose in agricultural organic waste is why it is difficult to degrade, and inoculating microbial agents capable of degrading lignocellulose is an important measure to promote recycling of agricultural organic waste. Microbial agents for degrading agricultural organic waste have been studied for decades, but most microbial agents for degrading lignocellulose, which are available on the market, have many problems in practical applications, such as easy inactivation of degrading microorganisms in a composting environment, antagonistic reaction of inoculated microorganisms with indigenous microorganisms in the heap.
Disclosure of Invention
In view of the above, the invention aims to provide a composite microbial agent which has stronger adaptability to the composting environment of agricultural organic waste, has stronger degradation capability and can accelerate the composting process of the agricultural organic waste.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a compound microbial agent, which comprises aspergillus niger, pseudo-pallidum, xylan bacillus, bacillus subtilis and amyloliquefaciens;
the volume ratio of aspergillus niger to pseudo-pallidum to xylan paenibacillus, bacillus subtilis to paenibacillus amyloliquefaciens is 1-2: 3 to 5:1 to 3: 4-8: 2 to 5.
Preferably, the total viable bacteria concentration of the compound microorganism bacterial agent is 2 multiplied by 10 8 ～2×10 10 cfu/ml。
The invention also provides a preparation method of the compound microbial agent, which comprises the following steps: inoculating each strain into a liquid culture medium, culturing until the strain becomes turbid to obtain each seed solution, culturing the obtained seed solution in an enrichment culture medium for 12-16 hours, and then, performing volume ratio of 1-2: 3 to 5:1 to 3: 4-8: and 2-5, mixing.
Preferably, the enrichment medium is beef extract peptone liquid medium and/or potato dextrose agar liquid medium.
Preferably, the culture conditions are those of 35-40℃and 220 rpm.
The invention also provides a method for degrading agricultural organic waste, which comprises the following steps: inoculating 1-3% of the composite microbial agent to each kilogram of compost material, and performing composting fermentation for 50-75 days.
Preferably, the water content of the compost material is 60-70% before the compost is fermented.
Preferably, the agricultural organic waste is agricultural straw.
The invention also provides application of the composite microbial agent in tomato straw composting fermentation.
Compared with the prior art, the invention has the following beneficial effects:
the composite microbial agent obtained by the invention can accelerate the composting process of agricultural organic waste. The comparison test shows that the pile added by the composite microbial agent is superior to the composting effect of the commercial microbial agent EM in the aspects of temperature, nutrient content, degradation rate of lignocellulose, enzyme activity and the like. The composite microbial agent obtained by the invention is more suitable for the heap environment, and antagonistic reaction does not exist between microbial strains, so that the antagonistic action between inoculated strains and original strains in the heap is reduced to a great extent. The composite microbial agent obtained by the invention has better promotion effect on agricultural organic waste composting.
Drawings
FIG. 1 is a graph showing the change of physicochemical properties of a pile under different treatments, wherein a to d respectively represent the changes of temperature, water content, pH and EC value in the composting process;
FIG. 2 is a graph showing the change of the lignocellulose content in the pile under different treatments, wherein a-c are the change of the cellulose, hemicellulose and lignin content in sequence;
FIG. 3 is a graph showing the change of organic matter content in the stack under different treatments;
FIG. 4 is a graph showing the change of ammonium nitrogen and nitrate nitrogen in a stack under different treatments, wherein a represents ammonium nitrogen and b represents nitrate nitrogen;
FIG. 5 is an X-ray diffraction analysis of the stack under various treatments;
FIG. 6 is a graph showing the change of enzyme activity of cellulase in the composting fermentation process under different treatments;
FIG. 7 is a graph showing the change in urease activity during the fermentation of compost under different treatments;
FIG. 8 is a graph showing the change in xylanase activity during compost fermentation under different treatments;
FIG. 9 is a graph showing the change in lignin peroxidase activity during compost fermentation under different treatments.
Detailed Description
The invention provides a compound microbial agent, which comprises aspergillus niger (Aspergillus niger), pallidum pseudopallidum (Falsochrobactrum ovis), paenibacillus xylan (paenibacillus xylolyticus), bacillus subtilis (bacillus subtilis) and paenibacillus amyloliquefaciens (Paenibacillus amylolyticus);
the preferred volume ratio of aspergillus niger, pseudo-pallidum, xylan bacillus, bacillus subtilis and bacillus amyloliquefaciens is 1-2: 3 to 5:1 to 3: 4-8: 2 to 5; more preferred mass ratio is 1:4:2:6:3. in the present invention, the Aspergillus niger, the Bacillus pseudopallidus, the Paenibacillus xylanolytic, the Bacillus subtilis and the Paenibacillus amyloliquefaciens are all purchased from conventional commercial products.
In the invention, the total viable bacteria concentration of the compound microorganism bacterial agent is preferably 2 multiplied by 10 8 ～2×10 10 cfu/ml; more preferably, the total viable bacteria concentration is 2×10 9 cfu/ml。
The invention also provides a preparation method of the compound microbial agent, which comprises the following steps: inoculating each strain into a liquid culture medium, culturing until the strain becomes turbid to obtain each seed solution, culturing the obtained seed solution in an enrichment culture medium for 12-16 hours, and then, performing volume ratio of 1-2: 3 to 5:1 to 3: 4-8: mixing 2-5; more preferably, the incubation time is 14 to 15 hours. The culture time of the invention can enable the strain to be cultured to the logarithmic phase, and the strain has the strongest growth capacity.
In the invention, the enrichment medium is beef extract peptone liquid medium and/or potato dextrose agar liquid medium. In the specific embodiment of the invention, the pseudo-pallidum, the paenibacillus xylan, the bacillus subtilis and the paenibacillus amyloliquefaciens are inoculated in a beef extract peptone liquid culture medium to be cultivated to a logarithmic phase; aspergillus niger is inoculated in potato dextrose agar liquid medium for culture until logarithmic phase. In the specific embodiment of the invention, the preparation method of the beef extract peptone liquid culture medium comprises the following steps: taking 10g/L peptone and 5g/L beef extract (NH) 4 ) 2 SO 4 2g/L of NaCl 5g/L, 1000mL of distilled water is added, the pH is regulated to 7.0+/-0.2 by NaOH, and sterilization treatment is carried out for 30 minutes under the condition that the set temperature of an autoclave is 121 ℃; the potato dextrose agar liquid medium is prepared as follows: taking 1L of potato juice (namely, 200g of peeled potatoes, cutting the peeled potatoes into small pieces, adding water to boil for 30 minutes, filtering out the potato pieces, adding distilled water to supplement the filtrate to 1L, and adjusting the pH to 7.2-7.4 with 20.0g/L of glucose). The sterilization treatment was also carried out at a temperature of 121℃in an autoclave for 30 minutes.
In the present invention, the culture conditions are preferably those of 35 to 40℃and 220 rpm; more preferably at 37℃and 220 rpm.
The invention also provides a method for degrading agricultural organic waste, which comprises the following steps: preferably inoculating 1-3% of the composite microbial agent to each kilogram of compost material, and fermenting the compost for 50-75 days; more preferably, 1.5 to 2 percent of the composite microbial agent is inoculated, and the compost is fermented for 65 to 70 days. The composite microbial agent disclosed by the invention can promote the degradation of agricultural organic waste, so that the weight loss rate of the agricultural organic waste after composting fermentation reaches more than 31.3%.
In the invention, the water content of the compost material is preferably 60-70% before composting fermentation; the water content is more preferably 65 to 68%.
In the invention, the agricultural organic waste is agricultural straw. In the present invention, the agricultural straw includes, but is not limited to, corn straw, rice straw, wheat straw, fruit and vegetable straw.
The invention also provides application of the composite microbial agent in tomato straw composting fermentation.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A compound microbial agent comprises Aspergillus niger, fructus Xanthii, paenibacillus xylan, bacillus subtilis, and Paenibacillus amyloliquefaciens;
the volume ratio of aspergillus niger to pseudo-pallidum to xylan bacillus, bacillus subtilis to amyloliquefaciens is 1:4:2:6:3.
example 2
A compound microbial agent comprises Aspergillus niger, fructus Xanthii, paenibacillus xylan, bacillus subtilis, and Paenibacillus amyloliquefaciens;
the volume ratio of aspergillus niger to pseudo-pallidum to xylan bacillus, bacillus subtilis to amyloliquefaciens is 2:5:1:8:5.
example 3
A compound microbial agent comprises Aspergillus niger, fructus Xanthii, paenibacillus xylan, bacillus subtilis, and Paenibacillus amyloliquefaciens;
the volume ratio of aspergillus niger to pseudo-pallidum to xylan bacillus, bacillus subtilis to amyloliquefaciens is 2:3:3:4:2.
example 4
The preparation method of the composite microbial agent comprises the following steps: inoculating Aspergillus niger, pseudo-pale bacillus, paecilomyces amyloliquefaciens, bacillus subtilis and Paecilomyces amyloliquefaciens into a liquid culture medium, culturing until the culture medium is turbid to obtain respective seed solutions, inoculating Aspergillus niger seed solution into a potato dextrose agar liquid culture medium, culturing for 16 hours to obtain Aspergillus niger culture solution, inoculating pseudo-pale bacillus, paecilomyces amyloliquefaciens, bacillus subtilis and Paecilomyces amyloliquefaciens seed solution into a beef extract peptone liquid culture medium, culturing for 14 hours to obtain each culture solution, diluting the Aspergillus niger, pseudo-pale bacillus, paecilomyces amyloliquefaciens, bacillus subtilis and Paecilomyces amyloliquefaciens culture solution to the same viable bacteria number, and mixing according to the volume ratio of the example 1 to obtain the composite microbial inoculum.
Example 5
A method of degrading agricultural organic waste comprising the steps of: 1% of the composite microbial agent described in the embodiment 1 is inoculated to each kilogram of corn stalks, and composting fermentation is carried out for 50 days.
Example 6
A method of degrading agricultural organic waste comprising the steps of: 1.5% of the composite microbial agent described in the embodiment 1 is inoculated to each kilogram of rice straw, and composting fermentation is carried out for 75 days.
Example 7
A method of degrading agricultural organic waste comprising the steps of: inoculating 2% of the composite microbial agent described in the embodiment 1 into each kilogram of wheat straw, and carrying out composting fermentation for 55 days.
Test example 1
The strain is optimally combined by adopting a uniform experimental design:
taking a 250mL conical flask, adding 1g of crushed 40-mesh straw powder, and adding solid fermentation ion liquid (solid fermentation ion liquid: ion mother liquid 0.1mL, NH) 4 NO 3 1 g，KH 2 PO 4 1.0 g，CaCl 2 0.1g, 1000mL of distilled water) 6mL, and autoclaving at 121℃for 15min. Inoculating the purified strain to a seed culture mediumAfter the medium culture, the medium culture was inoculated at a constant temperature of 40℃and 200rpm according to the inoculation ratio of Table 1. In order to meet the demands of microorganism growth metabolism on water and microelements, the water content should be controlled to be about 60% in the composting fermentation process, and small amount of ion mother liquor (ion mother liquor: mnSO) should be added into each conical flask every 3 days 4 ·7H 2 O 0.016g，ZnSO 4 ·7H 2 O 0.014g，CoCl 2 0.02 g，FeCl 3 0.1g, distilled water 100 mL). And taking out the corresponding conical flasks after fermentation, pouring the conical flasks into a weighed glass plate, and drying the glass plate in an oven until the weight is constant, so as to calculate the degradation rate.
TABLE 1 Strain proportioning table
| Uniform design | Aspergillus niger | Pseudo-pallidum | Paenibacillus xylan | Bacillus subtilis | Bacillus amyloliquefaciens |
| Factors of | x1 | x2 | x3 | x4 | x5 |
| N1 | 3 | 1 | 6 | 4 | 2 |
| N2 | 2 | 3 | 4 | 1 | 6 |
| N3 | 6 | 2 | 1 | 3 | 4 |
| N4 | 5 | 5 | 5 | 5 | 5 |
| N5 | 4 | 6 | 3 | 2 | 1 |
| N6 | 1 | 4 | 2 | 6 | 3 |
Table 2 straw weight loss test results
According to Table 2, when the inoculation ratio of Aspergillus niger, pseudo-pallidum, paenibacillus xylan, bacillus subtilis and Paenibacillus amyloliquefaciens is 1:4:2:6:3, the weight loss rate of the tomato straw after compost fermentation can reach more than 31.3%.
Test example 2
Composting experiments were performed at the Yang Ling experimental base of Shaanxi in 2022, 9 months to 11 months, with composting time lasting 65 days. The used materials are from tomato straws in greenhouse of facility vegetables near the experimental base, and the tomato straws are crushed into pieces of 1-4 cm after being aired. The experiment was carried out using a 60L composting barrel, and the dry weight of the composting material was about 15kg. Composting experiments are divided into three treatments, and a heap body without microbial inoculum is used as a control group (CK); taking 10% of the composite microbial agent obtained in inoculation example 4 as a ZS group; the EM group is inoculated with 10% of EM microbial agent. All treatments were adjusted to 60-70% water content by adding distilled water before composting. In order to ensure ventilation, the experiment adopts an indirect ventilation (ventilation is carried out once in 30 minutes) mode of an aeration pump to supply oxygen, and the ventilation quantity is 0.3L/min. Digital temperature sensors are installed at three positions of the compost tank (upper, middle and lower of the compost tank) and the temperature of the surrounding environment is recorded. In the composting period of the tomato straw, in order to ensure that the decomposition degree of the tomato straw in the composting barrel is as consistent as possible, manual turning is carried out during composting to mix the composting materials. Wherein the total viable bacteria concentration of the composite microbial agent and the EM microbial agent is 2 multiplied by 10 9 cfu/ml。
After each stack turning, samples were collected using a five-point sampling method, and a portion was stored at-20 ℃ for measurement of microbial and enzymatic activities. The other part is subjected to measurement including pH, EC, GI, water content, cellulose, hemicellulose, lignin, ammoniacal nitrogen, nitrate nitrogen and the like.
Analysis of results:
(1) The temperature is an important index for judging the maturity of the agricultural organic waste compost, and is a heating period, a thermophilic period, a cooling period and a decomposing period, which mainly influence the growth speed and activity of microorganisms in the compost, and further influence the decomposition rate of lignocellulose and the maturity of the compost. As can be seen from FIG. 1a, ZS enters the high temperature stage 2 days and 1 day earlier than EM and CK, respectively (temperature. Gtoreq.50℃), and each treatment ZS (63.81 ℃), EM (60.17 ℃) and CK (57.43 ℃) reaches the maximum value around day 8. Each treatment was maintained at the high temperature stage for 9 days (ZS), 7 days (EM) and 7 days (CK), respectively, to meet the standards for compost on temperature (compost temperature was maintained above 50 ℃ for more than 7 days). And then the temperature starts to drop, when the temperature of the pile body drops to about 25 ℃, the temperature of the pile body rises slightly, probably due to the fact that mesophilic microorganisms are reactively activated again, but the temperature is maintained to drop again for a plurality of days due to the lack of needed nutrients, the low ambient temperature and the like, and finally the temperature tends to be the ambient temperature until the composting is finished. The inoculation of ZS microbial inoculum increases the temperature of the pile (3-6 ℃ higher than other piles), and prolongs the high-temperature period of the compost (2 days longer than other treatments).
Moisture plays a key role in the microbial degradation of agricultural organic waste because it can participate in the metabolic activity of microorganisms only when the organic matter in the heap is water-soluble. In order to maintain the water content of the pile in a relatively proper range, the experiment adopts a composting barrel to perform composting, and only a small amount of water is released through a top cover exhaust valve of the composting barrel. The water content of each treatment at the beginning of composting is around 73%, and at day 3 the water content of each treatment is reduced, and then the water content is increased, and the treatment is in a dynamic stable state until the composting is finished (figure 1 b) due to the increase of the composting temperature and the fact that the added water does not permeate into the composting material. The reason why the compost moisture content is dynamically stable is as follows: condensing a large amount of water vapor generated at high temperature above the composting barrel, cooling and then reversing the water vapor to the composting body; a large amount of carbon dioxide and moisture are generated during the biodegradation of the organic matter. The reason for the smaller fluctuation of the water content in the CK treatment is probably due to the lower microbial activity in the stack, so that the stack has less water content due to the high Wen Sanshi and poorer organic substance decomposition capability.
As can be seen from fig. 1c, the trend of the pH change between treatments generally increases rapidly and then decreases slowly. The pH was raised to about 9.5 at about 6.7 at the initial stage of composting, but maintained at a pH of 7 to 9 which is favorable for microbial growth and metabolism throughout the composting process. The final pH is ZS (7.98) EM (8.14) CK (8.00) respectively, and the final pH meets composting standard (pH < 9).
The EC value represents the concentration of soluble salts in the compost, and the EC value of each treatment in the initial stage of the compost is about 4.3 ms/cm. As can be seen from FIG. 1d, as composting proceeds, the EC value gradually increases and then stabilizes, and the final EC value of ZS (5.9 ms/cm) is about 0.4ms/cm higher than that of EM (5.5 ms/cm) and CK (5.4 ms/cm). The ZS treatment is significantly higher than the other two treatments, probably due to the re-activation of mesophilic microorganisms in the heap as the temperature is reduced, again degrading the organic matter. In the cooling stage, a large amount of nitrate nitrogen is generated due to the reduction of the temperature, so that the concentration effect of the EC value of the stack is weakened. Until the composting is finished, the EC value of the pile body tends to be gentle.
(2) As can be seen from fig. 2a, the cellulose content in the initial ZS, EM and CK was 36.75%, 37.92% and 37.40%, respectively, and decreased to 19.61%, 21.59% and 23.28% after composting. From the figure, it is known that the cellulose degradation rate is fastest in 0-22 days of composting, and the degradation rate begins to slow down in 22-52 days and finally tends to be stable. The CK treated group cellulose degradation rate was relatively slow compared to ZS and EM, so inoculation with exogenous microbial agents was able to overstep cellulose degradation. The degradation capacity of the ZS and EM treatments on cellulose is not quite different, but the degradation capacity of the ZS treatment is stronger.
The degradation trend of hemicellulose in the compost is basically consistent with that of cellulose, and the hemicellulose has better thermal stability, so that the degradation rate of the hemicellulose is faster in the high-temperature stage of the compost. As can be seen from fig. 2b, the hemicellulose content was 9.42%, 9.715% and 9.85% at the initial stage of composting, respectively, and at the end of composting, the hemicellulose content was significantly reduced by 1.725%, 2.145% and 2.775% respectively. According to the curve, the degradation rate of hemicellulose is the fastest in days 0-14, and then the degradation rate begins to slow down, and the composting end is gradually flattened from 38 days until the composting end. In the degradation of hemicellulose, the trend of the treatments is almost uniform, probably due to the fact that hemicellulose is hybridized with cellulose, and when the cellulose is degraded, hemicellulose is hydrolyzed.
Lignin is composed of phenolic compounds, making it difficult to degrade by microorganisms, and lignin degradation products, mainly phenols and quinones, are important precursors for HS formation. As can be seen from fig. 2c, the relative lignin content rapidly increased between 0 and 22 days, each treatment reached a maximum of ZS (15.78%), EM (16.01%) and CK (15.12%) on day 38, followed by a slow start of the decrease, and finally lignin relative contents of ZS (14.87%), EM (13.93%) and CK (13.45%). The reason that the lignin content is not reduced and the lignin content is increased is that organic compounds such as protein, hemicellulose, cellulose and the like are more easily utilized by microorganisms in the early stage of composting, so that the degradation speed is high, and the relative lignin content in the early stage of composting is increased. As composting proceeds, some readily degradable materials are almost completely degraded, and lignin degradation rates are slower but faster than other organics. Thus, the relative lignin content started to decrease at day 38.
(3) As can be seen from fig. 3, as composting proceeds, microorganisms begin to degrade some recalcitrant substances, such as lignin and cellulose, which results in the organic matter beginning to slow down in the later stages of composting. Until the composting is finished, the content of organic matters reaches a minimum value ZS (370.97 g/kg) EM (390.96 g/kg) CK (398.05 g/kg). HA/FA is an important index for reacting the maturity of compost, and the ratio of HA/FA is continuously increased in the whole composting process. As the related degradation microorganisms are vigorously propagated in the early stage of composting, the conversion of FA to HA is promoted, and the HA/FA ratio is increased more rapidly. HA/FA of ZS (1.26) is higher than that of EM (1.19) and CK (1.12) until composting is finished, which shows that the addition of the microbial agent enhances the organic humification effect and promotes composting.
(4) As can be seen from fig. 4a, the trend of the ammoniacal nitrogen changes during composting is a trend of rising and then falling. All treatments reached peak ZS (5754.665 mg/kg), EM (5420.7 mg/kg) and CK (4340 mg/kg) around day 14. The ammoniacal nitrogen then begins to drop until the composting is completed. The final ammonia nitrogen content was ZS (550.4 mg/kg), EM (538.66 mg/kg) and CK (288.88 mg/kg), respectively. The increase in ammonia nitrogen content at the initial stage of composting may be due to the temperature and pH rise promoting mineralization of nitrogen compounds by microorganisms, promoting the production of ammonium in the heap and the volatilization of ammonia. The reason for the reduced ammonia nitrogen content at the later stages of composting may be: the excessive nitrogen element in the compost can be released in the form of ammonia gas; nitrifying bacteria.
As can be seen from fig. 4b, the presence of a significant amount of nitrate in the compost indicates that the compost is mature. The general trend of nitrate nitrogen throughout the composting process is rising until the nitrate nitrogen content of each treatment reaches a maximum ZS (2191.39 mg/kg), EM (1866.715 mg/kg) and CK (1486.475 mg/kg) at the end of composting. The nitrate nitrogen content tends to rise and then fall in 0-8 days of composting. Probably because the early composting temperature is not high, the activity of nitrifying bacteria is high. The reason for the decrease is that the nitrifying action of the heap is weakened because the activity of nitrifying bacteria is suppressed under thermophilic conditions.
(5) The compost samples were rinsed with distilled water and air dried at room temperature. The dried straw was ground to a powder and analyzed for crystallinity using an X-ray diffractometer. The continuous scanning angle is 10-80 degrees, the rated current is 40mA, and the rated voltage is 40KV;
the calculation formula of the crystallinity is: i CR ＝(I 1 -I 2 )/I 1 。
The crystallization index is proved to be one of the best predictors of the crystallization characteristics of cellulose, and the formation of a compact crystal structure in a cellulose crystallization area makes hydrolytic enzymes and water molecules difficult to invade and is a main reason for the difficulty in degrading lignocellulose in agricultural organic waste. As shown in FIG. 5, the crystallinity of each treatment after composting was reduced to a different extent than that of 0 CK. The maximum degree of crystallinity drop was 65ZS (10.25%), followed by 65EM (6.88%) and 65CK (6.23%).
The position of the X-ray diffraction peak is not changed basically between different treatments after composting treatment, but the intensity of each peak is changed to different degrees. The decrease in diffraction peak intensity at around 22 ° for each treatment compared to control group 0CK indicates that the crystalline structure of cellulose is destroyed, so that it can be demonstrated to some extent that the structure of cellulose is changed by degradation of the lignocellulose-degrading microorganisms involved in composting. The main reason for the reduced crystallinity of the compost material is that the crystalline structure of the cellulose is degraded into glucose absorbed by the microorganisms due to hydrolytic enzymes secreted by the relevant hydrolytic microorganisms in the heap. The degradation of agricultural organic waste can be promoted by inoculating microbial agents in the composting process of the agricultural organic waste through the reduction of crystallinity between different treatments. However, compared with 65EM treatment, the degradation effect of ZS is better, and microorganisms in ZS are more suitable for composting and degrading agricultural organic wastes to a certain extent.
(6) Enzyme activity determination:
as can be seen from fig. 6, the overall trend of cellulase activity was consistent. In the initial stage of composting, the cellulase activity of each treatment has an ascending trend. On day 8, the cellulase activities of ZS (1.59U/g), EM (0.77U/g) and CK (0.64U/g) all reach maximum values, mainly because the organic matters which are easy to degrade in the pile promote the reproduction of related microorganisms and promote the activity of the cellulase to be increased. Subsequently, the cellulase activity began to decline, ZS (0.48U/g), EM (0.35U/g) and CK (0.50U/g) and reached the enzyme activity minimum on day 26.
As can be seen from FIG. 7, ZS (2002.20U/g), EM (1845.63U/g), CK (1706.55U/g) urease activities were maintained at a high level at the initial stage of composting. As composting proceeds, the urease activity decreases dramatically, possibly due to the production of harmful substances inhibiting the urease activity by the microorganisms, and also due to the decrease in activity of the microorganisms, resulting in a decrease in enzyme yield. ZS (352.71U/g), EM (299.00U/g), CK (377.80U/g) urease activities were maintained at a low level until composting was completed, with little difference between treatments.
Xylanase activity is mainly the primary hydrolase involved in hemicellulose in agricultural organic waste. Xylan can be hydrolyzed to xylose and xylo-oligosaccharides. As can be seen from FIG. 8, xylanase activity was kept at a high level at the early stages of composting ZS (12.38U/g), EM (12.06U/g), CK (12.15U/g). As the xylanase activity gradually decreases as composting proceeds, ZS (4.50U/g), EM (3.73U/g), CK (3.68U/g) and xylanase activity are maintained at a lower level until the end of composting.
Lignin peroxidase can oxidize aromatic compounds of lignin to form aromatic free radicals, and the change of enzyme activity in the compost can reflect the degradation condition of lignin in agricultural organic waste in the compost. As can be seen from FIG. 9, ZS (5.32U/g), EM (5.21U/g), CK (5.61U/g) were maintained at a high level during the initial stage of composting. As composting proceeds, there appears to be a decrease in enzyme activity for each treatment on day 8, with the greatest decrease in EM (72.52%) and CK (76.79%) followed by ZS (25.75%). The reason why the ZS treatment enzyme activity was reduced less in magnitude is probably due to the increase in the number of heat-resistant microorganisms by the inoculated microbial agent.
In conclusion, the composite microbial agent obtained by the invention can accelerate the composting process of agricultural organic wastes. The pile added by the composite microbial agent is superior to the composting effect of the commercial microbial agent EM in the aspects of temperature, nutrient content, degradation rate of lignocellulose, enzyme activity and the like.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The composite microbial agent is characterized by comprising aspergillus niger, pseudo-pallidum, paenibacillus xylan, bacillus subtilis and paenibacillus amyloliquefaciens;
the volume ratio of aspergillus niger to pseudo-pallidum to xylan paenibacillus, bacillus subtilis to paenibacillus amyloliquefaciens is 1-2: 3 to 5:1 to 3: 4-8: 2 to 5.
2. The composite microbial agent according to claim 1, wherein the total viable bacteria concentration of the composite microbial agent is 2×10 8 ～2×10 10 cfu/ml。
3. The preparation method of the composite microbial agent as claimed in claim 1 or 2, which is characterized by comprising the following steps: inoculating each strain into a liquid culture medium, culturing until the strain becomes turbid to obtain each seed solution, culturing the obtained seed solution in an enrichment culture medium for 12-16 hours, and then, performing volume ratio of 1-2: 3 to 5:1 to 3: 4-8: and 2-5, mixing.
4. The method according to claim 3, wherein the enrichment medium is beef extract peptone liquid medium and/or potato dextrose agar liquid medium.
5. The method according to claim 3, wherein the culture conditions are those of 35 to 40℃and 220 rpm.
6. A method of degrading agricultural organic waste, comprising the steps of: inoculating 1-3% of the composite microbial agent in claim 1 or 2 to each kilogram of compost material, and fermenting the compost for 50-75 days.
7. The method of claim 6, wherein the compost material has a moisture content of 60 to 70%.
8. The method of claim 6, wherein the agricultural organic waste is agricultural straw.
9. The use of the composite microbial inoculant of claim 1 or 2 in tomato straw compost fermentation.
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