CN114317379A - Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof - Google Patents
Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN114317379A CN114317379A CN202210109040.4A CN202210109040A CN114317379A CN 114317379 A CN114317379 A CN 114317379A CN 202210109040 A CN202210109040 A CN 202210109040A CN 114317379 A CN114317379 A CN 114317379A
- Authority
- CN
- China
- Prior art keywords
- culture
- microbial inoculum
- parts
- cellulase
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of biological treatment, and provides a complex microbial inoculum for producing high-activity cellulase and a preparation method thereofMethods and uses. The compound microbial inoculum comprises the following microbial inoculum in parts by volume: 25-40 parts of bacillus licheniformis, 25-40 parts of paenibacillus jelly, 15-25 parts of bacillus subtilis and 15-25 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.0 multiplied by 109～1.5×109One per ml. The composite microbial inoculum can solve the problems of low decomposition speed of organic matters in the traditional septic tank, high cleaning frequency, increased sewage system load of effluent of the septic tank, operation requirement, maintenance cost and the like, can effectively decompose cellulose in water, reduces the amount of cellulose in the form of sludge in sewage treatment, and reduces the treatment load.
Description
Technical Field
The invention relates to the technical field of biological treatment, in particular to a complex microbial inoculum for producing high-activity cellulase, a preparation method and application thereof.
Background
The rural domestic sewage treatment is to remove and degrade harmful substances and polluted environment components in the domestic sewage for harmless treatment, wherein the septic tank is used as a traditional environment-friendly facility and plays an important role in the rural manure treatment process. However, the decomposition speed of organic matters in the septic tank is low, so that sludge is deposited at the bottom of the septic tank, the cleaning time of the septic tank is generally 3 months to one year, the cleaning frequency is low, the load of a sewage system for water outlet of the septic tank is increased, and the operation requirement and the maintenance cost are increased. Especially cellulose contained in the sewage of the septic tank brings great difficulty to the dispersion treatment of the sewage at the rear end, especially in rural areas, and is not beneficial to the dispersion treatment mode of the sewage in the rural areas. Therefore, a method for efficiently treating rural fecal sewage is urgently needed to be found so as to solve the problem of rural sewage treatment.
Disclosure of Invention
The invention aims to provide a composite microbial inoculum for producing high-activity cellulase, a preparation method and application thereof, the composite microbial inoculum can solve the problems of low decomposition speed of organic matters in the traditional septic tank, high cleaning frequency, increased sewage system load of effluent of the septic tank, operation requirement, maintenance cost and the like, can effectively decompose cellulose in water, reduces the amount of cellulose in a sludge form in sewage treatment, and reduces the treatment load.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a high-activity fertilizerThe cellulase compound microbial inoculum comprises the following microbial inoculum in parts by volume: 25-40 parts of bacillus licheniformis, 25-40 parts of paenibacillus jelly, 15-25 parts of bacillus subtilis and 15-25 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.0 multiplied by 109～1.5×109One per ml.
The invention also provides a preparation method of the composite microbial inoculum for producing the high-activity cellulase, which comprises the following steps:
(1) respectively activating and culturing bacillus licheniformis, bacillus mucilaginosus, bacillus subtilis and saccharomyces cerevisiae to obtain stock seeds;
(2) carrying out fermentation amplification culture on the stock obtained in the step (1) to obtain a zymophyte liquid;
(3) respectively inoculating the zymophyte liquid obtained in the step (2) into cellulase enrichment culture media for culture;
(4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
Further, the temperature of the activation culture in the step (1) is 28-32 ℃, the time of the activation culture is 24-48 h, and the oscillation frequency during the activation culture is 160-170 rpm.
Further, the inoculation volume ratio of the stock seeds in the fermentation and amplification culture in the step (2) is 1-5%, and the culture medium of the fermentation and amplification culture is an LB culture medium.
Further, the temperature of the fermentation amplification culture in the step (2) is 28-32 ℃, the stirring speed during the fermentation amplification culture is 100-150 r/min, the dissolved oxygen is 2-3 mg/L, and when the total number of the microorganisms in the zymophyte liquid reaches 1.0 multiplied by 109～1.5×109The fermentation was stopped at one/ml.
Further, the cellulase-enriched medium in the step (3) takes water as a solvent, and comprises the following components in concentration: 1-3 g/L of beef extract, 4-6 g/L of peptone, 22-28 g/L of cellulose powder, 1-2 g/L of ammonium sulfate, 0.5-1.5 g/L of monopotassium phosphate, 0.2-1 g/L of sodium chloride and 0.2-1 g/L of magnesium sulfate heptahydrate, wherein the pH value of the cellulase enrichment medium is 7.1-7.3.
Further, the temperature for culturing in the step (3) is 28-32 ℃, the time for culturing is 24-72 hours, and the oscillation frequency during culturing is 160-170 rpm.
The invention also provides an application of the composite microbial inoculum for producing the high-activity cellulase in degrading cellulose in rural excrement and sewage, wherein the usage amount of the composite microbial inoculum is 0.1-0.5% of the sewage mass.
The invention has the beneficial effects that:
(1) the composite microbial inoculum consists of a plurality of microorganisms, the activity of cellulase generated by fermentation for 24 hours is determined to be more than 4.2U/ml by the enzyme activity of filter paper, and the composite microbial inoculum has very high cellulase activity;
(2) the composite microbial inoculum can accelerate the decomposition of cellulose organic matters such as paper, fruit and vegetable residues, excrement residues and the like in the septic tank, prevent the blockage of the septic tank and reduce the frequency of cleaning the septic tank;
(3) the composite microbial inoculum can reduce the capacity of rural manure distributed treatment devices, reduce the facility investment of rural distributed sewage treatment, and has higher economic benefit, and after the sewage discharged from a septic tank enters a sewage treatment system, the amount of cellulose existing in the form of sludge in sewage treatment is reduced along with the decomposition of cellulose in the sewage, so that the generation amount of domestic sludge is reduced, and the composite microbial inoculum is particularly beneficial to the unattended state in the rural sewage treatment process;
(4) after the composite microbial inoculum is added into a septic tank, when the liquid dung fermented by the septic tank is used for farmland irrigation, the biological flora enters the soil, so that the excitation effect of microorganisms in the soil is further promoted, organic matters in the soil are utilized, the growth of crops is promoted, and the yield of grains, vegetables and the like is increased.
Drawings
FIG. 1 is a diagram showing the results of the activity measurement of cellulase produced by a complex microbial inoculum under different proportional conditions;
FIG. 2 is a graph for measuring cellulose degradation performance of the complex microbial inoculum under different proportion conditions.
Detailed Description
The invention provides a composite microbial inoculum for producing high-activity cellulase, which comprises the following microbial inoculum in parts by volume: 25-40 parts of bacillus licheniformis, 25-40 parts of bacillus mucilaginosus, 15-25 parts of bacillus subtilis and 15-25 parts of saccharomyces cerevisiae.
In the invention, the prepared composite microbial inoculum for producing the high-activity cellulase comprises 25-40 parts by volume of bacillus licheniformis, preferably 28-38 parts by volume, and more preferably 30-35 parts by volume.
According to the invention, the composite microbial inoculum for producing the high-activity cellulase comprises 25-40 parts by volume of paenibacillus jelly, preferably 28-38 parts by volume, and more preferably 30-35 parts by volume.
According to the invention, the composite microbial inoculum for producing the high-activity cellulase comprises 15-25 parts by volume of bacillus subtilis, preferably 18-23 parts by volume, and more preferably 19-21 parts by volume.
According to the invention, the composite microbial inoculum for producing the high-activity cellulase comprises 15-25 parts by volume of saccharomyces cerevisiae, preferably 18-23 parts by volume of saccharomyces cerevisiae, and more preferably 19-21 parts by volume of saccharomyces cerevisiae.
In the invention, the concentration of the bacteria in each microbial inoculum is 1.0 multiplied by 109～1.5×109One/ml, preferably 1.1X 109～1.4×109One/ml, more preferably 1.2X 109～1.3×109One per ml.
In the invention, the bacillus licheniformis, the paenibacillus mucilaginosus, the bacillus subtilis and the saccharomyces cerevisiae are all commercially available.
The invention also provides a preparation method of the composite microbial inoculum for producing the high-activity cellulase, which comprises the following steps:
(1) respectively activating and culturing bacillus licheniformis, bacillus mucilaginosus, bacillus subtilis and saccharomyces cerevisiae to obtain stock seeds;
(2) carrying out fermentation amplification culture on the stock obtained in the step (1) to obtain a zymophyte liquid;
(3) respectively inoculating the zymophyte liquid obtained in the step (2) into cellulase enrichment culture media for culture;
(4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
In the invention, bacillus licheniformis, paenibacillus jelly, bacillus subtilis and saccharomyces cerevisiae are respectively activated and cultured to obtain stock seeds.
In the present invention, the culture medium of the activation culture is preferably an LB medium; the temperature of the activation culture is preferably 28-32 ℃, and more preferably 29-31 ℃; the activation culture time is 24-48 h, preferably 30-45 h, and more preferably 32-40 h; the oscillation frequency during the activation culture is preferably 160-170 rpm, and more preferably 162-168 rpm.
The invention carries out fermentation and expanded culture after obtaining the stock. In the invention, the inoculation volume ratio of the stock seeds during the fermentation and scale-up culture is preferably 1-5%, and more preferably 2-4%; the culture medium for the fermentation amplification culture is preferably LB culture medium.
In the invention, the temperature of the fermentation amplification culture is preferably 28-32 ℃, and more preferably 29-31 ℃; the rotating speed of stirring during fermentation and amplification culture is 100-150 r/min, preferably 110-140 r/min, and more preferably 120-130 r/min; the dissolved oxygen in the culture medium is 2-3 mg/L, preferably 2.2-2.8 mg/L, and more preferably 2.4-2.6 mg/L; when the total number of the microorganisms in the zymophyte liquid reaches 1.0 multiplied by 109～1.5×109Stopping fermentation at one cell/ml, and preferably, the total number of bacteria is 1.1 × 109～1.4×109One/ml, more preferably 1.2X 109～1.3×109One per ml.
The invention respectively inoculates the zymophyte liquid in a cellulase enrichment culture medium for culture after obtaining the zymophyte liquid. In the invention, the cellulase-enriched culture medium takes water as a solvent and comprises the following components in concentration: 1-3 g/L beef extract, 4-6 g/L peptone, 22-28 g/L cellulose powder, 1-2 g/L ammonium sulfate, 0.5-1.5 g/L potassium dihydrogen phosphate, 0.2-1 g/L sodium chloride and 0.2-1 g/L magnesium sulfate heptahydrate.
According to the invention, the prepared cellulase-enriched culture medium comprises 1-3 g/L of beef extract, preferably 1.5-2.5 g/L, and more preferably 1.8-2.2 g/L.
In the invention, the cellulase-enriched culture medium comprises 4-6 g/L of peptone, preferably 4.5-5.5 g/L, and more preferably 4.8-5.2 g/L.
In the invention, the prepared cellulase-enriched culture medium comprises 22-28 g/L of cellulose powder, preferably 24-26 g/L, and more preferably 24.5-25.5 g/L.
In the invention, the cellulase-enriched culture medium comprises 1-2 g/L of ammonium sulfate, preferably 1.4-1.6 g/L, and more preferably 1.45-1.55 g/L.
In the invention, the cellulase-enriched culture medium comprises 0.5-1.5 g/L of monopotassium phosphate, preferably 0.8-1.2 g/L, and more preferably 0.9-1.1 g/L.
In the invention, the cellulase-enriched culture medium comprises 0.2-1 g/L of sodium chloride, preferably 0.4-0.8 g/L, and more preferably 0.5-0.6 g/L.
In the invention, the cellulase-enriched culture medium comprises 0.2-1 g/L magnesium sulfate heptahydrate, preferably 0.4-0.8 g/L, and more preferably 0.5-0.6 g/L.
In the invention, the pH value of the cellulase-enriched culture medium is preferably 7.1-7.3, and more preferably 7.15-7.25.
In the invention, the temperature of the culture is preferably 28-32 ℃, and more preferably 29-31 ℃; the culture time is 24-72 h, preferably 36-60 h, and more preferably 45-50 h; the shaking frequency during the culture is preferably 160-170 rpm, more preferably 162-168 rpm.
The invention also provides an application of the composite microbial inoculum for producing the high-activity cellulase in degrading cellulose in rural excrement and sewage, wherein the usage amount of the composite microbial inoculum is preferably 0.1-0.5%, more preferably 0.2-0.4%, and further preferably 0.3% of the sewage mass.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a complex microbial inoculum for producing high-activity cellulase, which comprises the following microbial inoculum in parts by volume: 40 parts of bacillus licheniformis, 40 parts of bacillus mucilaginosus and bacillus subtilis20 parts of bacillus and 20 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.0 multiplied by 109One per ml. The preparation method of the complex microbial inoculum comprises the following steps: (1) respectively activating and culturing bacillus licheniformis, paenibacillus jelly, bacillus subtilis and saccharomyces cerevisiae, wherein the specific steps of the activation culture are as follows: putting an ampoule bottle which is stored in a refrigerator at the temperature of-80 ℃ and is filled with dry powder strains in the refrigerator at the temperature of-20 ℃ for 24 hours; wiping the outer tube with cotton dipped with 70% alcohol, and heating the tip of the outer tube on the flame; thirdly, dropping a plurality of drops of sterile water at the heating position to break the outer tube, and then breaking the tip by using tweezers; and fourthly, adding 1ml of culture solution into the ampoule tube, and adding the culture solution into the sterilized LB culture solution for activation culture after the solid in the ampoule tube is dissolved. The temperature of the activation culture is 30 ℃, the time of the activation culture is 36h, and the oscillation frequency during the activation culture is 160rpm, so as to obtain an original seed; (2) performing fermentation amplification culture on the stock obtained in the step (1), wherein the inoculation volume ratio of the stock during the fermentation amplification culture is 3%, the culture medium for the fermentation amplification culture is an LB culture medium, the temperature for the fermentation amplification culture is 28 ℃, the stirring speed during the fermentation amplification culture is 120r/min, the dissolved oxygen is 2mg/L, and the total microbial count in a zymophyte liquid reaches 1 × 109Collecting zymocyte liquid when the strain per ml is detected; (3) respectively inoculating the zymophyte liquid obtained in the step (2) into a cellulase enrichment culture medium for culture, wherein the cellulase enrichment culture medium takes water as a solvent and comprises the following components in concentration: 1g/L beef extract, 4g/L peptone, 22g/L cellulose powder, 1g/L ammonium sulfate, 0.5g/L monopotassium phosphate, 0.2g/L sodium chloride and 0.2g/L magnesium sulfate heptahydrate, wherein the pH value of the cellulase enrichment medium is 7.1; the culture temperature is 28 ℃, the culture time is 48h, and the oscillation frequency is 160rpm during culture; (4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
Example 2
The embodiment provides a complex microbial inoculum for producing high-activity cellulase, which comprises the following microbial inoculum in parts by volume: 30 parts of bacillus licheniformis, 35 parts of paenibacillus jelly, 15 parts of bacillus subtilis and 20 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.3 multiplied by 109One per ml.The preparation method of the complex microbial inoculum comprises the following steps: (1) respectively activating and culturing bacillus licheniformis, paenibacillus jelly, bacillus subtilis and saccharomyces cerevisiae, wherein the specific steps of the activation culture are as follows: putting an ampoule bottle which is stored in a refrigerator at the temperature of-80 ℃ and is filled with dry powder strains in the refrigerator at the temperature of-20 ℃ for 24 hours; wiping the outer tube with cotton dipped with 70% alcohol, and heating the tip of the outer tube on the flame; thirdly, dropping a plurality of drops of sterile water at the heating position to break the outer tube, and then breaking the tip by using tweezers; and fourthly, adding 1ml of culture solution into the ampoule tube, and adding the culture solution into the sterilized LB culture solution for activation culture after the solid in the ampoule tube is dissolved. The temperature of the activation culture is 28 ℃, the time of the activation culture is 24h, and the oscillation frequency during the activation culture is 165pm, so that stock seeds are obtained; (2) performing fermentation amplification culture on the stock obtained in the step (1), wherein the inoculation volume ratio of the stock during the fermentation amplification culture is 2%, the culture medium for the fermentation amplification culture is an LB culture medium, the temperature for the fermentation amplification culture is 30 ℃, the stirring speed during the fermentation amplification culture is 100r/min, the dissolved oxygen is 2.5mg/L, and the total microbial count in a zymophyte liquid reaches 1.2 multiplied by 109Collecting zymocyte liquid when the strain per ml is detected; (3) respectively inoculating the zymophyte liquid obtained in the step (2) into a cellulase enrichment culture medium for culture, wherein the cellulase enrichment culture medium takes water as a solvent and comprises the following components in concentration: 2g/L of beef extract, 5g/L of peptone, 25g/L of cellulose powder, 1.5g/L of ammonium sulfate, 1.0g/L of monopotassium phosphate, 0.5g/L of sodium chloride and 0.5g/L of magnesium sulfate heptahydrate, wherein the pH value of the cellulase enrichment medium is 7.2; the culture temperature is 30 ℃, the culture time is 36h, and the shaking frequency is 165rpm during culture; (4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
Example 3
The embodiment provides a complex microbial inoculum for producing high-activity cellulase, which comprises the following microbial inoculum in parts by volume: 35 parts of bacillus licheniformis, 30 parts of paenibacillus jelly, 20 parts of bacillus subtilis and 15 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.5 multiplied by 109One per ml. The preparation method of the complex microbial inoculum comprises the following steps: (1) mixing Bacillus licheniformis and jellyRespectively activating and culturing Paenibacillus semenii, Bacillus subtilis and Saccharomyces cerevisiae, wherein the activating and culturing specifically comprises the following steps: putting an ampoule bottle which is stored in a refrigerator at the temperature of-80 ℃ and is filled with dry powder strains in the refrigerator at the temperature of-20 ℃ for 24 hours; wiping the outer tube with cotton dipped with 70% alcohol, and heating the tip of the outer tube on the flame; thirdly, dropping a plurality of drops of sterile water at the heating position to break the outer tube, and then breaking the tip by using tweezers; and fourthly, adding 1ml of culture solution into the ampoule tube, and adding the culture solution into the sterilized LB culture solution for activation culture after the solid in the ampoule tube is dissolved. The temperature of the activation culture is 32 ℃, the time of the activation culture is 48h, and the oscillation frequency during the activation culture is 170rpm, so as to obtain an original seed; (2) performing fermentation amplification culture on the stock obtained in the step (1), wherein the inoculation volume ratio of the stock during the fermentation amplification culture is 5%, the culture medium for the fermentation amplification culture is an LB culture medium, the temperature for the fermentation amplification culture is 32 ℃, the stirring speed during the fermentation amplification culture is 150r/min, the dissolved oxygen is 3mg/L, and the total microbial count in a zymophyte liquid reaches 1.5 multiplied by 109Collecting zymocyte liquid when the strain per ml is detected; (3) respectively inoculating the zymophyte liquid obtained in the step (2) into a cellulase enrichment culture medium for culture, wherein the cellulase enrichment culture medium takes water as a solvent and comprises the following components in concentration: 3g/L of beef extract, 6g/L of peptone, 28g/L of cellulose powder, 2g/L of ammonium sulfate, 1.5g/L of monopotassium phosphate, 1g/L of sodium chloride and 1g/L of magnesium sulfate heptahydrate, wherein the pH value of the cellulase enrichment medium is 7.3; the culture temperature is 32 ℃, the culture time is 72h, and the oscillation frequency is 170rpm during culture; (4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
Experimental example 1 Single-Strain organic matter degradation experiment
Respectively inoculating four single strains of bacillus licheniformis, bacillus mucilaginosus, bacillus subtilis and saccharomyces cerevisiae with high cellulase activity into a cellulase enrichment culture medium, performing shake culture at 37 ℃ for 24 hours, respectively inoculating the four single strains into triangular flasks filled with 100mL of septic tank bottom mud according to the inoculation amount of 5%, performing 6 parallels on each sample, performing shake culture at 30 ℃ and 160r/min, respectively, taking samples for 2, 3 and 4 days, taking 2 parallels of each sample for measuring MLSS, calculating the average value, and inoculating the cellulose enrichment culture solution without strains with the same volume in a control group. The MLSS of the initial sludge was 2832mg/L, and the results are shown in Table 1:
table 1: degradation of organic matter by single strain
As can be seen from Table 1, compared with the control group, the single strain can produce better organic matter decomposition effect after being added, wherein the bacillus licheniformis has the best effect. In the organic matter decomposition process, the removal rate is increased firstly and then reduced, the reason for analyzing the organic matter decomposition process is that the microorganisms can grow by utilizing nutrient substances in a water sample within 1 day of initial culture, the nutrient substances are exhausted in the second day, the microorganisms begin to enter a decline period from a stable period of growth, most of the microorganisms are in the decline period in the third day, thalli are subjected to autolysis, and some endogenous hydrolase is released to degrade macromolecular components of cell walls and cell membranes, so that the structure of activated sludge flocs is damaged. The destruction of the floc structure allows the enzyme produced by these microorganisms to permeate into the aged flocs, resulting in further disintegration of the structure, thereby producing more small molecular substances to be utilized by the microorganisms, and producing more biological enzymes again, thereby achieving the effect of organic matter degradation, but as the new microorganisms utilize nutrients, the increased amount of the microorganisms can form new flocs, but the total amount of activated sludge is reduced.
Experimental example 2 degradation experiment of organic matters in complex microbial inoculum
Respectively inoculating the strains into cellulase enrichment culture medium, performing shake culture at 37 ℃ for 24h, and then performing volume ratio V (Bacillus licheniformis): v (paenibacillus jelly): v (bacillus subtilis): v (saccharomyces cerevisiae), and the mixed materials are inoculated into a triangular flask filled with 100mL of bottom mud taken from a septic tank, the decomposition effect of organic matters is detected, the initial MLSS of the organic matters is 2682mg/L, and the method is shown in the table 2:
table 2: decomposition of organic matters by composite strains under different proportioning conditions
As can be seen from Table 2, the compound ratio is 2:2:1:1, and when the organic matter is cultured for 3 days, the decomposition effect of the organic matter is the best, and is as high as 13.91 percent, which is better than that of any single strain. The fibrous matter contained in the organic matter of the bottom mud is decomposed fast under the action of cellulase, and the extracellular matter of the floc in the bottom mud mainly consists of hydrocarbon and protein, so the floc structure is destroyed after the thalli autolyze, and the substances are utilized by microorganisms. The degradation effect is also reduced in the fourth day, because microorganisms grow up by using the degradation products, the organic matters are increased, but the overall organic matter degradation effect is very obvious.
Experimental example 3 cellulase production Activity measurement
Four single strains of bacillus licheniformis, paenibacillus jelly, bacillus subtilis and saccharomyces cerevisiae with high cellulase production activity are mixed according to the volume ratio of 1:1:1:1, 1:2:1:2, 2:1:2:1, 1:1:2:2 and 2:2:1:1, then cultured for 10 days, and sampled every day from the next day and the cellulase activity of the mixed strain is measured, and the measurement result is shown in figure 1. As can be seen from the figure, the cellulase activity produced by the composite microbial inoculum is more than 4.88U/ml, and the composite microbial inoculum can produce a large amount of cellulase.
Experimental example 4 cellulose degradation Properties measurement
Filter paper disintegration medium: (NH)4)2SO41.0g/L，MgSO4·7H2O 0.5g/L，KH2PO41.0g/L, 0.1g/L yeast extract, 1/4 pieces of filter paper/triangular flask, pH 7.0; the filter paper strip disintegration experiment can reflect the comprehensive enzyme activity level of various cellulases secreted by different strains. Respectively inoculating the obtained four single bacterial suspensions of the bacillus licheniformis, the paenibacillus mucilaginosus, the bacillus subtilis and the saccharomyces cerevisiae into a 250ml triangular flask filled with 100ml of filter paper strip disintegration culture medium after mixing according to the volume ratio of 1:1:1:1, 1:2:1:2, 2:1:2:1, 1:1:2:2 and 2:2:1:1In the medium, shake cultivation (30 ℃, 130r/min), the disintegration of the filter paper strips is regularly observed, the measurement results are shown in figure 2, and blank control groups (inoculated with culture solution without fungicide in the same volume), inoculation volume ratio of 1:1:1:1, inoculation volume ratio of 1:2:1:2, inoculation volume ratio of 2:1:2:1, inoculation volume ratio of 1:1:2:2 and inoculation volume ratio of 2:2:1:1 are sequentially arranged from left to right in the figure.
Comparing cellulase activity determination and filter paper disintegration experiments, the composite microbial inoculum obtained by mixing the strains of bacillus licheniformis, bacillus mucilaginosus, bacillus subtilis and saccharomyces cerevisiae according to a certain proportion has a good effect on degradation of organic matters, and corresponds to a plurality of strains with higher cellulase activity, and the composite microbial inoculum mixed according to a certain volume ratio can also produce a large amount of cellulase.
According to the embodiment, the composite microbial inoculum for producing the high-activity cellulase and the preparation method and application thereof are provided, the composite microbial inoculum can solve the problems of low decomposition speed of organic matters in the traditional septic tank, high cleaning frequency, increased load of a sewage system for effluent of the septic tank, operation requirement, maintenance cost and the like, can effectively decompose cellulose in water, reduces the amount of the cellulose in the form of sludge in sewage treatment, and reduces the treatment load.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The composite microbial inoculum for producing the high-activity cellulase is characterized by comprising the following microbial inocula in parts by volume: 25-40 parts of bacillus licheniformis, 25-40 parts of paenibacillus jelly, 15-25 parts of bacillus subtilis and 15-25 parts of saccharomyces cerevisiae; the concentration of the bacteria in each microbial inoculum is 1.0 multiplied by 109～1.5×109One per ml.
2. The method for preparing the complex microbial inoculum of claim 1, which is characterized by comprising the following steps:
(1) respectively activating and culturing bacillus licheniformis, bacillus mucilaginosus, bacillus subtilis and saccharomyces cerevisiae to obtain stock seeds;
(2) carrying out fermentation amplification culture on the stock obtained in the step (1) to obtain a zymophyte liquid;
(3) respectively inoculating the zymophyte liquid obtained in the step (2) into cellulase enrichment culture media for culture;
(4) and (4) mixing the bacterial liquid obtained in the step (3) to obtain the composite microbial inoculum for producing the high-activity cellulase.
3. The preparation method according to claim 2, wherein the temperature of the activation culture in the step (1) is 28-32 ℃, the time of the activation culture is 24-48 h, and the shaking frequency during the activation culture is 160-170 rpm.
4. The method according to claim 2, wherein the inoculation volume ratio of the stock in the step (2) is 1-5% during the fermentation and scale-up culture, and the culture medium of the fermentation and scale-up culture is LB culture medium.
5. The method according to claim 2, wherein the temperature of the fermentation amplification culture in the step (2) is 28 to 32 ℃, the rotation speed of stirring during the fermentation amplification culture is 100 to 150r/min, the dissolved oxygen is 2 to 3mg/L, and when the total number of the microorganisms in the fermentation broth reaches 1.0X 109～1.5×109The fermentation was stopped at one/ml.
6. The method according to claim 2, wherein the cellulase-enriched medium in the step (3) is water as a solvent, and comprises the following components in the following concentrations: 1-3 g/L of beef extract, 4-6 g/L of peptone, 22-28 g/L of cellulose powder, 1-2 g/L of ammonium sulfate, 0.5-1.5 g/L of monopotassium phosphate, 0.2-1 g/L of sodium chloride and 0.2-1 g/L of magnesium sulfate heptahydrate, wherein the pH value of the cellulase enrichment medium is 7.1-7.3.
7. The method according to claim 2 or 6, wherein the temperature of the culture in step (3) is 28-32 ℃, the time of the culture is 24-72 hours, and the shaking frequency during the culture is 160-170 rpm.
8. The application of the composite bacterial agent in degrading cellulose in rural fecal sewage of claim 1, wherein the usage amount of the composite bacterial agent is 0.1-0.5% of the sewage quality.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210109040.4A CN114317379A (en) | 2022-01-28 | 2022-01-28 | Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210109040.4A CN114317379A (en) | 2022-01-28 | 2022-01-28 | Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114317379A true CN114317379A (en) | 2022-04-12 |
Family
ID=81030369
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210109040.4A Pending CN114317379A (en) | 2022-01-28 | 2022-01-28 | Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114317379A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275906A (en) * | 2013-06-08 | 2013-09-04 | 福建卫生职业技术学院 | Compound microbial fermentation inoculant and its application |
| CN104388341A (en) * | 2014-10-29 | 2015-03-04 | 青岛明月蓝海生物科技有限公司 | Bacillus mucilaginosus strain and application thereof in seaweed degradation |
| CN105039210A (en) * | 2015-06-29 | 2015-11-11 | 北京沃土天地生物科技有限公司 | Complex lively bacterial preparation as well as water soluble fertilizer and application thereof |
| CN105543136A (en) * | 2016-01-11 | 2016-05-04 | 鹤壁市人元生物技术发展有限公司 | Bio-organic fertilizer fermented liquid microbial agent and preparing method thereof |
| CN107285953A (en) * | 2017-06-29 | 2017-10-24 | 重庆市万源禽蛋食品有限公司 | Composite zymocyte, biological organic fertilizer and preparation method thereof |
| CN110438024A (en) * | 2019-04-17 | 2019-11-12 | 浙江亲水园生物科技有限公司 | A kind of bacillus subtilis strain and its application |
| CN110893318A (en) * | 2019-12-07 | 2020-03-20 | 湖南科美洁环保科技有限公司 | Preparation method and use method of public toilet microbial deodorant |
-
2022
- 2022-01-28 CN CN202210109040.4A patent/CN114317379A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103275906A (en) * | 2013-06-08 | 2013-09-04 | 福建卫生职业技术学院 | Compound microbial fermentation inoculant and its application |
| CN104388341A (en) * | 2014-10-29 | 2015-03-04 | 青岛明月蓝海生物科技有限公司 | Bacillus mucilaginosus strain and application thereof in seaweed degradation |
| CN105039210A (en) * | 2015-06-29 | 2015-11-11 | 北京沃土天地生物科技有限公司 | Complex lively bacterial preparation as well as water soluble fertilizer and application thereof |
| CN105543136A (en) * | 2016-01-11 | 2016-05-04 | 鹤壁市人元生物技术发展有限公司 | Bio-organic fertilizer fermented liquid microbial agent and preparing method thereof |
| CN107285953A (en) * | 2017-06-29 | 2017-10-24 | 重庆市万源禽蛋食品有限公司 | Composite zymocyte, biological organic fertilizer and preparation method thereof |
| CN110438024A (en) * | 2019-04-17 | 2019-11-12 | 浙江亲水园生物科技有限公司 | A kind of bacillus subtilis strain and its application |
| CN110893318A (en) * | 2019-12-07 | 2020-03-20 | 湖南科美洁环保科技有限公司 | Preparation method and use method of public toilet microbial deodorant |
Non-Patent Citations (2)
| Title |
|---|
| 崔保安等: "《动物微生物学 第3版》", 中国农业出版社, pages: 75 * |
| 王世英等: "保氮钝重微生物菌剂的创制及其在生物有机肥中的应用", 《科技成果》, pages 1 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109182193B (en) | Microbial agent and preparation method and application thereof | |
| CN101486969B (en) | Preparation of compost fermentation complex bacterial agent | |
| CN106399209B (en) | Composite microbial inoculum for degrading high-grease kitchen waste and preparation method thereof | |
| CN109022327B (en) | Preparation method of microbial mixed inoculant and application of microbial mixed inoculant in high-temperature composting | |
| CN101244955B (en) | Biological fertilizer and manufacture method thereof | |
| CN102093975B (en) | Composite fungicide for rapid degradation of organic waste and applications thereof | |
| CN104293694A (en) | Preparation method for sludge aerobic composting composite inoculum | |
| CN117603888B (en) | Bacillus cereus and application thereof in cultivation tail water treatment | |
| CN101215532B (en) | Bacillus megaterium and its application and application method in ferment bacteria | |
| CN113321548B (en) | Organic fertilizer prepared by comprehensively utilizing wastes generated in beer production and preparation method thereof | |
| CN114561327A (en) | Cellulose degradation composite microbial inoculum and preparation method and application thereof | |
| CN107285482B (en) | Environment-friendly enzyme for purifying eutrophic water and preparation method thereof | |
| CN109576189A (en) | A kind of black and odorous water composite bacteria agent and its preparation method and application | |
| CN111154661B (en) | Complex microbial inoculant and application thereof | |
| CN105645596A (en) | Active sludge preprocessing agent, preparation method and application method thereof | |
| CN112795516A (en) | Complex microbial inoculant for decomposing and fermenting chicken manure compost | |
| CN112625948A (en) | Bacillus tequilensis S1 with nitrogen fixation function and application thereof in compost | |
| CN110272853B (en) | Microbial culture method | |
| CN114317379A (en) | Complex microbial inoculant for producing high-activity cellulase as well as preparation method and application thereof | |
| CN110452852B (en) | Farmer market biomass waste fermentation inoculant and preparation method thereof | |
| CN110240284B (en) | Microbial koji block for sewage treatment, preparation method and application thereof | |
| CN110803947A (en) | Manufacturing method for producing efficient water flush fertilizer by utilizing biogas slurry | |
| CN114230019B (en) | Application of probiotics activated sludge in sewage treatment | |
| CN113717882B (en) | Geobacillus galactose BWTGW1.1 and application thereof | |
| CN108913616B (en) | Method for preparing biological bacterial fertilizer by utilizing tobacco leaf residues |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |