CN108913616B - Method for preparing biological bacterial fertilizer by utilizing tobacco leaf residues - Google Patents
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Abstract
The invention discloses a method for preparing biological bacterial fertilizer by utilizing tobacco leaf residues. Taking the residue after extracting active ingredients from dry tobacco leaves or fresh tobacco leaves as a culture medium, adding seed solutions of bacillus subtilis, bacillus mucilaginosus and paecilomyces lilacinus, uniformly mixing, and performing solid culture for 7-15 days to obtain corresponding biological bacterial fertilizer, wherein the number of spores is more than 109cfu/g. The method takes the tobacco leaf residues after the effective components are extracted as the solid culture medium, and other nutrient substances are not required to be added, so that the production cost is saved; the microorganism in the product exists in the form of spore, and can be stably stored for a long time, thereby ensuring the microbial activity of the product. The product is rich in beneficial microorganisms and organic matters, can improve the content of organic matters in soil, improve the micro-ecological environment of the soil, reduce the using amount of chemical fertilizers, reduce plant diseases and insect pests, and improve the quality and the yield of crops.
Description
Technical Field
The invention belongs to the technical field of biological agriculture, relates to preparation of a composite biological bacterial fertilizer, and relates to comprehensive utilization of waste tobacco leaf residues.
Background
Tobacco production in China is all over the country, the tobacco yield per year is 400-500 ten thousand tons, the annual total amount of tobacco waste is 90-100 ten thousand tons, and the tobacco waste accounts for about 18-25% of the total yield. In addition, the number of field fresh tobacco leaves discarded due to improper climate, pest and disease damage or improper management or unqualified field fresh tobacco leaves is also extremely large, so the comprehensive utilization of tobacco waste becomes one of important research directions in the domestic tobacco industry. The tobacco leaves are rich in protein, saccharides, lignin, cellulose, organic acid, pectin and the like, the content of nitrogen, phosphorus and potassium is obviously higher than that of straws of grain crops, and particularly, the total nitrogen is 1.5-3%, the total sugar is 10-20%, and the total potassium is 2-5%. The tobacco leaf residue after extracting effective components (such as nicotine, solanesol and the like) is still rich in organic matters and other trace elements, is a good substrate for solid culture of microorganisms, and the development of a simple and feasible microorganism culture process is urgently needed.
The solid culture refers to the process of microorganism growth and metabolism on a culture medium almost without free water, and has the advantages of simple equipment, low energy consumption, easy operation, simple post-treatment, little pollution, basically no wastewater discharge, simple culture medium and the like. With the discovery of novel excellent strains and the application of gene detection technology, the conditions of solid state culture are gradually improved, and the culture of single strains tends to be mixed strain culture. The biological bacterial fertilizer usually comprises a plurality of microorganisms, and commonly used strains comprise bacillus subtilis, bacillus mucilaginosus, bacillus licheniformis, bacillus megaterium, side chain bacillus brevis, bacillus amyloliquefaciens, paecilomyces lilacinus and the like.
From published documents in recent years, it can be found that the following methods are mainly used for producing the biological bacterial fertilizer by using various cheap biomass resources such as agricultural and forestry wastes (such as straws, edible fungus residues, corncobs and the like), industrial waste biomass (such as residues in sugar and wine making), animal wastes and the like as raw materials: 1) the biomass and the microbial inoculum are simply mixed, such as CN 107673936A, CN 107721713A and the like. 2) The biomass is decomposed by composting, and then a plurality of functional microorganisms such as CN 107746356A, CN 106336269A, CN 106187590A and the like are added for mixing. 3) Two-stage solid culture: composting decomposed biomass, and adding functional microorganism such as CN 106007824A for solid culture. 4) Solid culture is carried out by taking biomass as a substrate, such as CN 107337517A, CN 107417457A and the like.
In the prior art, some reports of biological bacterial fertilizers of tobacco leaves exist, and the utilized tobacco leaves are mainly waste and inferior tobacco leaves or fresh tobacco leaves discarded in the field, such as CN103274785A, CN 105175193A, CN 103274784A and the like. Wherein, the active substances of the tobacco leaves are not extracted, which greatly causes resource waste. The adopted production process mainly comprises composting, the formula is complex, a lot of auxiliary materials need to be added, the consumed time is long, and the production process usually needs 1-2 months. The common problems with this way of producing compost are: the interaction, species and strain number change of microorganisms in the production process are unclear, and the final strain number and proportion are difficult to control effectively. Therefore, in the field of comprehensive utilization of tobacco leaves, there is a need to develop a technology that can be embedded in the existing tobacco industry technology framework to further develop the application of raw materials.
In conclusion, the tobacco leaf biological bacterial fertilizer is developed according to the current utilization situation of the waste tobacco leaves, the growth characteristics of the tobacco leaves and the production situation of the microbial bacterial fertilizer, the production process is simple, the number and the proportion of each bacterium are clear, and the tobacco leaf biological bacterial fertilizer has important significance for the utilization of the waste tobacco leaves and the development and the utilization of the biological bacterial fertilizer in the tobacco industry.
Disclosure of Invention
The invention aims to provide a technical scheme for fully utilizing tobacco leaf residues after active substances are extracted. The method is a method for preparing biological bacterial fertilizer by using the waste tobacco leaf residue of an active product industrial chain to carry out mixed bacteria solid culture.
In order to realize the aim, the invention discloses a method for preparing biological bacterial manure by utilizing tobacco leaf residues, which comprises the steps of taking the tobacco leaf residues as a solid culture medium, inoculating mixed strains and carrying out solid culture; wherein the mixed strain is a mixture of Bacillus subtilis, Bacillus mucilaginosus and Paecilomyces lilacinus.
The method takes the waste tobacco leaf residues as raw materials, inoculates seed liquid of bacillus subtilis, bacillus mucilaginosus and paecilomyces lilacinus, carries out mixed culture, improves the composition proportion of N, P, organic matters and other substances, obtains the composite microbial fertilizer, and realizes high-valued comprehensive utilization of tobacco waste.
Compared with the prior art, the invention has the following advantages:
firstly, the raw materials are wide in source and low in cost. The invention completely adopts the waste tobacco leaves and water as culture raw materials, has wide sources and low price, does not need complex pretreatment, utilizes the strain fermentation to convert the waste tobacco leaves into an important biomass resource, effectively utilizes active substances such as starch, organic acid, protein and the like in the waste tobacco leaves, and is rich in C, N, P and trace elements such as K, Ca, Mg and the like.
And secondly, the production cost is low. The solid culture technology adopted by the invention has the advantages of simple equipment, low energy consumption, easy operation, simple post-treatment, less pollution, basically no wastewater discharge and simple culture medium; the selected strains can normally grow at normal temperature of more than 10 ℃, strict temperature control and humidity control are not needed, and the equipment investment is greatly simplified.
Thirdly, the sense smell is good. The raw material of the invention is waste tobacco leaves which have strong tobacco fragrance, meanwhile, in the culture process, bacillus subtilis generates spice substances such as acetoin and the like, and a light cream fragrance is mixed in the strong tobacco fragrance; the paecilomyces lilacinus can produce lilac villous mycelium, so that the product has better color and luster sense.
Fourthly, multiple strains are mixed and cultured, and the culture process is simplified. The three kinds of bacteria are simultaneously inoculated into the tobacco leaf culture medium, mixed and cultured in the tobacco leaves, and interact to form a stable micro-ecological environment, which is favorable for the survival and growth of the three kinds of bacteria in the soil. By adjusting the inoculation ratios of different bacteria, products with different strain ratios can be obtained finally, and the effective control of the quantity and the ratio of the strains is realized. Moreover, the content of N in the fermented bacterial manure is obviously increased, and the quality of the bacterial manure is further improved.
Drawings
FIG. 1 is a graph showing the change in viable count of three types of bacteria when they are mixed and cultured at an indoor temperature of 15 to 25 ℃.
FIG. 2 is a graph showing the change in the number of spores of three types of bacteria when they are mixed and cultured at an indoor temperature of 15 ℃ to 25 ℃.
FIG. 3 is a graph showing the change in viable cell count of three types of bacteria when they are mixed and cultured in an incubator at 30 ℃.
FIG. 4 is a graph showing the change in the number of spores of three kinds of bacteria when they were mixed and cultured in an incubator at 30 ℃.
Detailed Description
The technical scheme of the invention comprises the steps of taking tobacco leaf residues as a solid culture medium, inoculating mixed strains and carrying out solid culture; wherein the mixed strain is a mixture of Bacillus subtilis, Bacillus mucilaginosus and Paecilomyces lilacinus.
Specifically, in the mixed strain, the ratio of bacillus mucilaginosus, bacillus subtilis and paecilomyces lilacinus is 1-10: 1 according to the number of viable bacteria.
More specifically, the mixed strain can be obtained by mixing seed culture solutions of three microorganisms; the preparation method of the seed culture solution comprises the following steps: inoculating the three kinds of microorganisms into seed culture medium, respectively, shake culturing at 150-200 r/min and 30-37 deg.C to OD620Is 4-6, wherein:
the seed culture medium of the bacillus mucilaginosus is 5-50 times of diluent, preferably 10-30 times of diluent, of molasses containing 0.2-1 g/100ml of ammonium phosphate;
the bacillus subtilis seed culture medium is 5-50 times of dilution liquid of molasses, and preferably 10-30 times of dilution liquid;
the paecilomyces lilacinus seed culture medium is 5-50 times of dilution of molasses, and preferably 10-30 times of dilution.
On the other hand, in the technical means of the present invention, the microbial fermentation is characteristically performed using tobacco leaf residue. The residue of the tobacco leaves is the residual part of the tobacco leaves after active substances such as nicotine, solanesol, chlorogenic acid and the like are removed by extraction and organic solvents are removed. In a specific embodiment, the tobacco leaf residue is the residue of tobacco leaves after active substance extraction, and the content of nicotine, solanesol and chlorogenic acid in the tobacco leaf residue is not more than 0.5mg/g, not more than 0.2mg/g and not more than 0.5mg/g based on the weight of dry residue. The removal of these active substances can be carried out by a method described in the art, for example, the method described in CN 104086425A. The invention provides a specific extraction technical scheme, which is characterized in that active substances in tobacco leaves are extracted, and the residual tobacco leaf residues are used for fermentation of the biological bacterial manure, wherein the method comprises the following steps: mixing the crushed dry tobacco leaves with 90% (volume concentration) ethanol water solution according to the mass ratio of the material liquid of 1: 5-10, carrying out heat reflux treatment at 50-80 ℃ for 0.5 hour, repeating for 1-5 times, removing ethanol from the extracted residues, and drying. More specifically, the method for obtaining the tobacco leaf residue comprises the following steps: adding 1L 90% ethanol into 100g dry tobacco leaf, thermally refluxing at 50 deg.C for 30min, filtering, separating tobacco leaf from extractive solution, and continuously thermally refluxing for 3 times, each time for 30 min. And then, drying the tobacco leaf residues, namely the tobacco leaf residues used in the embodiment of the invention, which can be used for preparing a tobacco leaf solid culture medium. The residual active substances in the obtained dry tobacco leaf residue are measured by the following method: taking 2g of dry tobacco leaf residue, adding 2mL of ethanol solution (70%), performing ultrasonic treatment for 30min, centrifuging, and taking supernatant for liquid chromatography detection of nicotine and chlorogenic acid. Taking 2g of dry tobacco leaf residues, adding 5mL of absolute ethyl alcohol, carrying out ultrasonic treatment for 30min, centrifuging, taking supernate, and using the supernate for detecting solanesol by liquid chromatography. Calculated by HPLC data, each gram of dry tobacco leaf residue contains 164.71 mug of nicotine, 41.25 mug of solanesol and 173.22 mug of chlorogenic acid after being treated by the extracted active substances.
In another specific embodiment of the invention, the solid culture medium is a mixture of tobacco leaf residues and water according to a mass ratio of 1: 1.2-2.0. Preferably 1: 1.4-1.6. The inoculation amount of the mixed strain is 1-15% of the total mass of the solid culture medium.
In another specific embodiment, the solid-state culture is carried out for 7-15 days, the material is turned over once every 24-48 hours, and the culture temperature is 10-37 ℃, preferably 20-30 ℃.
The present invention is further described below in connection with non-limiting examples which will enable one of ordinary skill in the art to more fully understand the present invention, but which are not intended to limit the scope of the present invention in any way.
Unless otherwise specified, the species used in the present invention are as follows:
b, bacillus subtilis: inoculating Bacillus subtilis strain (CGMCC13141 and Bacillus subtilis microbial inoculum respectively purchased from Qingdao root-source organism, Shandong green-ridge organism, Beihaiqiangxing organism, Jiangxi spring organism and Deqiang organism) into tobacco solid culture medium for culturing and domesticating, screening out the strain with vigorous growth, finding no obvious antagonism through a plate antagonism experiment, and preserving the strain for later use after mixed culture of LB culture medium.
B, Bacillus mucilaginosus: inoculating Bacillus mucilaginosus strain (purchased from kindling organisms) into a tobacco leaf solid culture medium for culture and domestication, screening out a strain with vigorous growth, and preserving the strain for later use after the culture of an LB culture medium.
Paecilomyces lilacinus: inoculating Bacillus mucilaginosus strain (purchased from kindling organisms) into a tobacco leaf solid culture medium for culture and domestication, screening out a strain with vigorous growth, and preserving the strain for later use after PDA culture medium culture.
The composition of the medium used in this specification is as follows:
LB medium (1L): 10g of peptone, 5g of yeast powder and 10g of NaCl. The pH was adjusted to 7.0 with NaOH and autoclaved at 121 ℃ for 20 min.
PDA culture medium: 200g of potato is boiled in water, filtered by eight layers of gauze, added with 20g of glucose, cooled, added with water to complement to 1L, and sterilized for 20min at 115 ℃. .
Bacillus subtilis seed medium (1L): 33.3g of molasses (with the sugar content of 42-50 percent) is added with water to 1L and sterilized for 20min at 115 ℃.
Bacillus mucilaginosus seed medium (1L): 33.3g of molasses (with the sugar content of 42-50 percent) and 4g of ammonium phosphate, adding water to 1L, and sterilizing at 115 ℃ for 20 min.
Paecilomyces lilacinus seed culture medium (1L): 33.3g of molasses (with the sugar content of 42-50 percent) is added with water to 1L and sterilized for 20min at 115 ℃.
The activation method of the strains in the specification is as follows:
activation of bacillus subtilis: inoculating 1mL of strain stored in refrigerator at-70 deg.C on clean bench into 100mLLB culture medium, shake culturing at 37 deg.C for 20 hr at 200r/min, OD 6204 to 6.
Activation of bacillus mucilaginosus: inoculating 1mL of strain stored in refrigerator at-70 deg.C on clean bench into 100mLLB culture medium, shake culturing at 30 deg.C for 20 hr at 200r/min, OD 6204 to 6.
Activation of paecilomyces lilacinus: storing in a refrigerator at-70 deg.C on a clean benchInoculating 1mL of the strain in 100mLPDA culture medium, performing shake culture at 30 ℃ for 20h at 200r/min, and performing OD 6204 to 6.
The analytical methods used in this specification are as follows:
measurement of cell Density (OD)620): in liquid culture, absorbance was measured at 620nm using a supernatant from which the cells were removed by centrifugation as a control.
The number of viable bacteria in the sample was measured by dilution coating plate method: according to a detection method (5.1 effective viable count) of effective viable counts in the microbial fertilizer product inspection regulations (NY 2321-2013).
Determination of spore (spore) number: weighing a solid fermentation sample, adding water, mixing uniformly, heating in a water bath at 80 ℃ for 20min, standing for 20min, and carrying out subsequent operations according to a detection method of effective viable bacteria number (5.1 effective viable bacteria number) in the microbial fertilizer product inspection regulations (NY 2321-2013).
Example 1
Preparation of microbial seed solutions
(1) Inoculating 1mL of Bacillus subtilis solution which is activated and cultured in LB culture medium for 20h to 100mL of Bacillus subtilis seed culture medium for amplification culture, and performing shake culture at 37 ℃ at 200r/min for 20h and OD620And obtaining the bacillus subtilis seed solution when the concentration reaches 4-6.
(2) Inoculating 1mL of Bacillus mucilaginosus solution which is activated and cultured in LB culture medium for 20h to 100mL of Bacillus mucilaginosus seed culture medium for amplification culture, and performing shake culture at 30 ℃ at 200r/min for 20h and OD620And obtaining the bacillus mucilaginosus seed liquid when the yield reaches 4-6.
(3) Inoculating 1mL of Paecilomyces lilacinus bacterial solution which is activated and cultured in a PDA culture medium for 20h to 100mL of Paecilomyces lilacinus seed culture medium for amplification culture, and performing shake culture at 30 ℃ at 200r/min for 20h, wherein OD is620And reaching 4-6, namely the paecilomyces lilacinus seed solution.
(4) Mixing the three seed solutions according to a certain proportion to obtain the mixed strain seed solution.
Example 2
Experiment for influence of tobacco raw materials on thallus growth
(1) 60g of dried tobacco leaf residues are taken and mixed in a 500mL triangular flask according to the feed-liquid ratio of the residues to water of 1:1.8 (mass ratio) to prepare the tobacco leaf culture medium. Inoculating a bacillus subtilis seed solution with the total mass of 10%, and statically culturing for 4 days in a constant temperature box with the temperature of 37 ℃, and turning the materials once every 48 hours. The viable count and the spore count were counted in plate culture. The viable count and the spore count are respectively 2.5 × 1013cfu/g and 5X 1010cfu/g。
(2) Crushing untreated dry tobacco leaves to be used as a tobacco leaf culture medium raw material, and fermenting the bacillus subtilis according to the method in the step (1). The culture time is 5 days and 7 days, and the obtained viable count is 2.15 × 108cfu/g and 6.37X 108cfu/g, number of spores 4.68X 108cfu/g and 8.45X 108cfu/g。
Therefore, the pretreated tobacco leaf residues are used as fermentation raw materials, the viable count is higher, and the expected culture effect can be achieved in a shorter period more easily.
Example 3
Comparison of Single and Complex species cultures
(1) 35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating a bacillus subtilis seed solution with the total mass of 10%, and performing static culture in a constant temperature box with the temperature of 30 ℃, and turning the materials once every 48 hours. The number of viable bacteria and the number of spores both reached the maximum value at day 10, wherein the number of viable bacteria was 7X 1013cfu/g, number of spores 1.5X 1013cfu/g。
(2) 35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating bacillus mucilaginosus seed solution with the total mass of 10%, and standing and culturing in a constant temperature box with the temperature of 30 ℃, and turning the material once every 48 hours. The number of viable bacteria and the number of spores both reached the maximum at day 12, wherein the number of viable bacteria was 2X 1013cfu/g, number of spores 1.6X 1013cfu/g。
(3) 35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating the paecilomyces lilacinus seed solution with the total mass of 10%, and standing and culturing in a constant temperature box at 30 ℃ and turning over once every 48 h. The number of live bacteria and spores are both reached on day 10Maximum value, in which the number of viable bacteria is 1X 1014cfu/g, number of spores 2X 1013cfu/g。
(4) 35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating mixed strain seed liquid (the proportion of the three strains is 1:1:1) with the total mass of 10 percent, and standing and culturing in a constant temperature box at the temperature of 30 ℃ and turning over once every 48 hours. The total number of viable bacteria reached a maximum at day 8, amounting to 1.5X 1015cfu/g, the total number of spores reached 2.5X 10 on day 1011cfu/g, the culture time was prolonged and the number of spores continued to increase.
Example 4
Experiment of influence of inoculation ratio on growth of complex bacteria
35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. 8.4mL of mixed bacteria seed liquid is inoculated, wherein the ratio of viable count of the Bacillus mucilaginosus to the Bacillus subtilis to the Paecilomyces lilacinus in the solid culture medium is 1:1:1, 3:1:2 and 6:1: 3. Standing and culturing in a constant temperature box at 30 ℃ and turning the materials once every 48 hours. The number of live bacteria and the number of spores are counted by plate culture, and the three bacteria are distinguished by morphological differences. The total number of viable bacteria reached a maximum at day 8, which was 1.5X 10 respectively15cfu/g、4.5×1015cfu/g and 5.2X 1014cfu/g, wherein the ratio of the three bacteria is 2.7:1:6.1, 6.8:1:6.8 and 3.0:1:1.3 respectively. The total number of spores at day 13 was 3.42X 10, respectively11cfu/g、1.8×1012cfu/g and 6.9X 1012cfu/g, wherein the ratio of the three bacteria is 2.7:1:2.4, 1.4:1:1.1 and 1.6:1:1.1 respectively.
Example 5
Preparation of mixed bacterial manure under indoor temperature condition
(1) 35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. 8.4mL of mixed bacteria seed liquid is inoculated, wherein the ratio of viable count to Bacillus mucilaginosus to the ratio of Bacillus subtilis to Paecilomyces lilacinus in the solid culture medium is 6: 3: 1. Standing and culturing at the indoor temperature of 15-25 ℃ and turning the materials once every 48 hours. The number of live bacteria and the number of spores are counted by plate culture, and the three bacteria are distinguished by morphological differences. The growth curves of viable count and spore count are shown in FIG. 1 and FIG. 1 respectivelyAs shown in fig. 2. The biomass of the microorganism is gradually increased in 0-8 days, the viable count of the microorganism is gradually reduced after reaching the maximum value in 8 days, and the maximum value can reach 1014cfu/g. The first 8 days produced fewer spores, followed by a rapid increase in the number of spores of bacillus mucilaginosus. By day 11, the number of spores of each strain reached 1011cfu/g. The ratio of spores of the three bacteria on day 13 is 1.7: 0.7: 1. Drying and bagging to obtain the biological bacterial fertilizer.
(2) 80g of the dried tobacco leaf residue and 112mL of water were put in a 500mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating 19.2mL of mixed bacteria seed liquid, wherein the ratio of viable count is bacillus mucilaginosus: b, bacillus subtilis: paecilomyces lilacinus (6: 3: 1). Standing and culturing at the indoor temperature of 15-20 ℃, and turning the materials once every 48 hours. The number of live bacteria and the number of spores are counted by plate culture, and the three bacteria are distinguished by morphological differences. Viable cell count and spore count growth curves are shown in fig. 3 and 4, respectively. Culturing for 10 days until the number of spores of the three bacteria reaches 109cfu/g, ratio of bacillus mucilaginosus: b, bacillus subtilis: paecilomyces lilacinus is 3: 1.9: 1. Drying and bagging to obtain the microbial compound bacterial fertilizer.
Example 6
Regulation and control of bacterial manure pH by mixed bacterial culture
35g of the dried tobacco leaf residue and 49mL of water were placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Respectively inoculating bacillus subtilis seed solution and mixed seed solution (the proportion of the three bacteria is 1:1:1) with the total mass of 10 percent, standing and culturing in a constant temperature box at the temperature of 30 ℃, turning over once every 48 hours, sampling 0.3g, measuring the pH value, and mixing according to the proportion of 1: 10 (sample: water) and purified water (pH 6.48), stirring well, standing for 30min, measuring with pH meter, recording after reading is stable, and paralleling three groups of each sample. In the initial stage of thallus culture, the solid-state culture condition is weakly acidic, the pH gradually increases along with the progress of thallus growth and metabolism, wherein the pH of the bacillus subtilis single-thallus culture medium is obviously increased, the maximum value is reached within 72 hours and is about 9, and then the pH fluctuates between 8.5 and 9.5; the pH value of the mixed culture medium gradually increases 7 days before, and keeps stable at about 8-8.5 at 8 days. Under the condition of no external substance, the mixed culture more effectively controls the pH value of the product.
Example 7
Influence of residual ethanol in dry tobacco leaves on solid culture of mixed bacteria
35g of dry tobacco leaf residue and 49mL of water (containing 1%, 2% and 3% ethanol by volume) are placed in a 250mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating 8.4mL of mixed bacteria seed liquid, wherein the ratio of viable count is bacillus mucilaginosus: b, bacillus subtilis: paecilomyces lilacinus (6: 3: 1). Standing and culturing at the indoor temperature of 8-15 ℃, and turning the materials once every 48 hours. The number of live bacteria and the number of spores are counted by plate culture, and the three bacteria are distinguished by morphological differences. The spore number of the three bacteria can reach 10 after 8 days of culture6cfu/g。
Example 8
Mixed bacterial fertilizer prepared from fresh tobacco leaf residues
20g of fresh tobacco leaf residue and 40mL of water are put into a 500mL conical flask and sterilized at 115 ℃ for 20 min. Inoculating 6mL of mixed strain seed liquid, wherein the ratio of viable count is Bacillus mucilaginosus: b, bacillus subtilis: paecilomyces lilacinus (6: 3: 1). Standing and culturing at indoor temperature (8-15 ℃) and turning the materials once every 48 hours. The number of live bacteria and the number of spores are counted by plate culture, and the three bacteria are distinguished by morphological differences. Culturing for 7 days until the number of spores of only Bacillus subtilis reaches 109cfu/g。
Claims (5)
1. A method for preparing biological bacterial manure by utilizing tobacco leaf residues is characterized by comprising the step of taking the tobacco leaf residues as a solid culture medium, inoculating mixed strains and carrying out solid culture; wherein the solid culture medium is a mixture of tobacco leaf residues and water according to the mass ratio of 1: 1.2-2.0, and the mixed strain is bacillus subtilis (Bacillus subtilis)Bacillus subtilis ) Bacillus mucilaginosus (B), (B)Bacillus mucilaginosus ) And paecilomyces lilacinus (A. lilacinus) ((B.))Paecilomyces lilacinus ) A mixture of (a);
in the mixed strain, the ratio of the bacillus mucilaginosus, the bacillus subtilis and the paecilomyces lilacinus is 1-10: 1 according to the number of viable bacteria;
the mixed strain is obtained by mixing seed culture solutions of three microorganisms;
the preparation method of the seed culture solution comprises the following steps: inoculating the three kinds of microorganisms into seed culture medium, respectively, shake culturing at 150-200 r/min and 30-37 deg.C to OD620Is 4-6, wherein:
the seed culture medium of the bacillus mucilaginosus is 5-50 times of dilution liquid of molasses containing 0.2-1 g/100ml of ammonium phosphate;
the bacillus subtilis seed culture medium is 5-50 times of dilution liquid of molasses;
the paecilomyces lilacinus seed culture medium is 5-50 times of dilution of molasses.
2. The method as claimed in claim 1, wherein the amount of the mixed strain is 1-15% of the total mass of the solid medium.
3. The method according to claim 2, wherein the solid culture is carried out for 7-15 days and is carried out at a temperature of 10-37 ℃ with stirring every 24-48 h.
4. The method according to claim 2, wherein the tobacco leaf residue is the residue of tobacco leaves after active substance extraction, and the content of nicotine, solanesol and chlorogenic acid in the tobacco leaf residue is not more than 0.5mg/g, not more than 0.2mg/g and not more than 0.5mg/g based on the weight of dry residue.
5. A process according to claim 4, wherein the tobacco leaf residue is prepared by: mixing the crushed dry tobacco leaves with 90% ethanol water solution according to the mass ratio of 1: 5-10, carrying out hot reflux treatment at 50-80 ℃ for 0.5 hour, repeating for 1-5 times, removing ethanol from the extracted residues, and drying.
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