CN116004405A - Culture method of Hansenula polymorpha - Google Patents
Culture method of Hansenula polymorpha Download PDFInfo
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- CN116004405A CN116004405A CN202310159542.2A CN202310159542A CN116004405A CN 116004405 A CN116004405 A CN 116004405A CN 202310159542 A CN202310159542 A CN 202310159542A CN 116004405 A CN116004405 A CN 116004405A
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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
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/02—Odour removal or prevention of malodour
Abstract
The invention provides a method for culturing hansenula polymorpha, and particularly relates to the technical field of microbial preparations. The culture method provided by the invention adopts solid culture, and has lower equipment requirement; the raw material cost is low, and the production cost of 1 ton is below 200 yuan; the hansenula polymorpha culture obtained by the culture can effectively reduce the release amount of malodorous gases such as ammonia, hydrogen sulfide and the like in the livestock manure; in addition, the odor component is decomposed, and meanwhile, pleasant fragrance is generated, so that the odor masking effect is achieved.
Description
The application is a divisional application of patent application named as culture method of Hansenula cerealis and microbial deodorization method of livestock and poultry manure, and the application date of the original application is 2019, 12, 18 and 201911308923.2.
Technical Field
The invention relates to the technical field of microbial preparations, in particular to a method for culturing Hansenula polymorpha.
Background
The large-scale and intensive development of livestock and poultry breeding industry promotes the development of agricultural economy and simultaneously brings serious environmental pollution, wherein malodorous smell generated in the process of discharging and stacking livestock and poultry manure has bad influence on human beings, animals and surrounding environment. The odor components in the livestock and poultry manure comprise: volatile sulfur compounds, nitrogen compounds, indoles and phenols and volatile fatty acids are mainly derived from the catabolism of certain components of the feces by microorganisms in the feces.
At present, the deodorization of livestock and poultry manure mainly comprises three types of physical methods, chemical methods and biological methods. The physical method is to use an adsorbent and a masking agent to adsorb and mask to realize deodorization, but the cost is high. The chemical method is to add chemical substances to react with malodorous substances to achieve the deodorizing effect, such as oxidizing the reductive malodorous substances into harmless odorless substances by using a strong oxidant; the calcium superphosphate reacts with ammonia gas generated in the manure, so that the ammonia gas concentration is reduced; the substances such as potassium permanganate, copper sulfate, acetic acid and the like are utilized to spray the padding, kill and inhibit the activities of microorganisms, and inhibit and reduce the generation of harmful gases. The chemical method has the defects of complex reaction, high cost, difficult recycling of excrement, and the like, and is difficult to popularize. The biological method is to degrade malodorous components into odorless and harmless final products by utilizing the metabolism of microorganisms, achieves the aim of deodorization, has the advantages of high efficiency, no secondary pollution, convenient management and the like, and is a main method for deodorizing the livestock and poultry manure.
At present, various microorganism strains are used for deodorizing feces, and common biological strains include bacillus, nitrifying bacteria, denitrifying bacteria, saccharomycetes, photosynthetic bacteria, lactic acid bacteria, aspergillus niger and the like. However, these strains have poor deodorizing effect, and the production and culture of the strains generally use liquid culture, and the required equipment (e.g., fermenter) and components of the culture medium cause high production costs.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for culturing hansenula polymorpha, which adopts solid culture and has lower equipment requirement; the raw material cost is low, and the production cost of 1 ton is below 200 yuan; the hansenula polymorpha culture obtained by the culture can effectively reduce the release amount of malodorous gases such as ammonia, hydrogen sulfide and the like in the livestock manure; in addition, the odor component is decomposed, and meanwhile, pleasant fragrance is generated, so that the odor masking effect is achieved.
In order to achieve the above object, the present invention provides the following solutions:
a method for culturing Hansenula polymorpha comprises the following steps:
(1) Inoculating Hansenula polymorpha in molasses liquid culture medium for culture to obtain liquid culture solution; the culture is shake culture of a shaking table; the temperature of the culture is 28-32 ℃ and the humidity is 70-85%; the culture time is 16-20 h;
(2) Inoculating the liquid culture solution on a solid culture medium for culture to obtain a hansenula polymorpha solid culture; the solid culture medium is prepared from raw materials including sugarcane skin, pineapple skin and ginkgo leaf; the mass ratio of the volume of the liquid culture solution to the solid culture medium is 5-8 mL:100g; the thickness of the solid culture medium is 10-15 cm; the concentration of Hansenula polymorpha in the liquid culture solution is (2-5) multiplied by 10 7 cfu/mL; the temperature of the culture is 28-32 ℃ and the humidity is 70-85%; the culture time is 48-60 hours; the preparation of the solid culture medium comprises the following steps:
(a) Drying sugarcane skin, pineapple skin and ginkgo leaf to obtain dry matters; the mass ratio of the sugarcane rind to the pineapple rind to the ginkgo leaf is 2-4: 2 to 5:3 to 6; the moisture content of the dry matter is less than 15%;
(b) Crushing and mixing the dry matters to obtain a mixture;
(c) Mixing the mixture with water to obtain a solid culture medium; the water content of the solid culture medium is 45-60%;
the effective viable count of the yeast solid culture is 2.2X10 by calculation of plate viable count 9 CFU/g～3.1×10 9 CFU/g。
Preferably, the method further comprises:
culturing Hansenula polymorpha to obtain a solid Hansenula polymorpha culture;
mixing the solid culture of Hansenula polymorpha with livestock manure to be treated, and stacking; the mass ratio of the solid culture of the Hansenula polymorpha to the livestock manure to be treated is 1-2: 50; the thickness of the pile is 30-40 cm; the stacking time is 48 hours; and turning the materials for 3-4 times during stacking.
Preferably, after the turning, the odor disappearance time is 36 to 48 hours.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects:
the invention provides a method for culturing hansenula polymorpha, which comprises the following steps: (1) Inoculating Hansenula polymorpha in molasses liquid culture medium for culture to obtain culture solution; (2) Inoculating the liquid culture solution on a solid culture medium prepared from raw materials including sugarcane skin, pineapple skin and ginkgo leaf, and culturing to obtain the hansenula cerealis solid culture. The culture method provided by the invention adopts solid culture, the equipment requirement is lower, the raw materials of the solid culture medium are waste, sugar is provided for the growth of bacteria by the sugarcane skin and pineapple skin, protein is provided for the growth of bacteria by the ginkgo leaf residue, the waste is effectively utilized, the culture cost is lower, and the yeast can grow and reproduce rapidly on the solid culture medium to reach higher bacteria number. Furthermore, hansenula polymorpha cultured by the method for culturing Hansenula polymorpha provided by the invention can decompose odor components and generate pleasant fragrance. The deodorizing agent is applied to deodorizing livestock and poultry manure, and can effectively reduce the release amount of malodorous gases such as ammonia, hydrogen sulfide and the like in the livestock and poultry manure; while decomposing the odor component, a pleasant fragrance is generated, and the effect of masking the odor is achieved.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The inclusion and inclusion of "and" having "and any variations thereof in the description and claims of the present application are intended to cover a non-exclusive inclusion. For example, inclusion of a list of steps, processes, methods, etc. is not limited to the listed steps but may alternatively include steps not listed or may alternatively include other steps inherent to such processes, methods, products, or apparatus.
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
The invention provides a method for culturing hansenula polymorpha, which comprises the following steps: (1) Inoculating Hansenula polymorpha in molasses liquid culture medium for culture to obtain culture solution; (2) Inoculating the liquid culture solution on a solid culture medium for culture to obtain a hansenula polymorpha solid culture; the solid culture medium is prepared from raw materials including sugarcane skin, pineapple skin and ginkgo leaf.
The invention inoculates Hansenula cerealis in molasses liquid culture medium for culture to obtain culture liquid. The source of the Hansenula polymorpha is not particularly limited, and the Hansenula polymorpha is preferably purchased from China general microbiological culture Collection center. In the invention, the molasses liquid medium comprises the following components: sugar, ammonium sulfate and water; the culture is preferably shake culture of a shaking table; the temperature of the culture is preferably 28 to 32 ℃, and more preferably 29 to 31 ℃; the humidity is preferably 70 to 85%, more preferably 75 to 80%; the time of the culture is preferably 16 to 20 hours, more preferably 17 to 19 hours. The molasses liquid culture medium can fully meet the growth of the Hansenula polymorpha, and the growth of the Hansenula polymorpha can be promoted in a proper culture environment and time.
After the culture solution is obtained, the liquid culture solution is inoculated on a solid culture medium for culture, so that the solid culture of Hansenula polymorpha is obtained. In the invention, the solid culture medium is prepared from raw materials including sugarcane rind, pineapple rind and ginkgo leaf; the mass ratio of the sugarcane rind to the pineapple rind to the ginkgo leaf is preferably 2-4: 2 to 5:3 to 6. The sources of the sugarcane rind, the pineapple rind and the ginkgo leaf are not particularly limited; can be the waste residues of sugarcane skin, pineapple skin and ginkgo leaf. The invention adopts solid culture, has lower equipment requirement, and the raw materials of the solid culture medium are waste, so the cost is low; sugar is provided for the growth of bacteria by the sugarcane rind and the pineapple rind, protein is provided for the growth of bacteria by the ginkgo leaf residue, waste is effectively utilized, the culture cost is low, and yeast can grow and propagate rapidly on the solid culture medium to achieve higher bacteria number.
In the present invention, the preparation of the solid medium preferably includes the steps of: drying sugarcane skin, pineapple skin and ginkgo leaf to obtain dry matters. The drying method of the present invention is not particularly limited, and a conventional drying method in the art may be used. The moisture content of the dry matter is preferably less than 15%. The invention can reduce the pollution rate of mixed bacteria after drying the sugarcane skin, pineapple skin and ginkgo leaves, thereby providing more sufficient nutrition and space for the growth of Hansenula polymorpha.
After the dry matter is obtained, the dry matter is crushed and mixed to obtain a mixture. The mixing method of the present invention is not particularly limited, and a mixing method conventional in the art may be used.
After the mixture is obtained, the mixture is mixed with water to obtain a solid culture medium. The moisture content of the solid medium is preferably 45 to 60%, more preferably 50 to 55%. The addition of suitable water can promote the growth and metabolism of Hansenula polymorpha.
In the invention, the mass ratio of the volume of the liquid culture solution to the solid culture medium is preferably 5-8 mL:100g, more preferably 6 to 7mL:100g; the thickness of the solid medium is preferably 10 to 15cm, more preferably 12 to 13cm; the concentration of Hansenula polymorpha in the culture solution is preferably (2-5) x 10 7 cfu/mL. The temperature of the culture is preferably 28 to 32 ℃, and more preferably 29 to 31 ℃; the humidity is preferably 70 to 85%, more preferably 75 to 80%; the time for the culture is preferably 48 to 60 hours, more preferably 50 to 58 hours. The proper volume of the liquid culture solution and the mass ratio of the solid culture medium enable the growth metabolism of the hansenula polymorpha to be accelerated, and the solid fermentation environment can promote the generation of extracellular substances of the hansenula polymorpha, so that the deodorizing effect is achieved.
The concentration of the hansenula polymorpha in the hansenula polymorpha solid fermentation product obtained by adopting the culture method of the invention reaches (2.2-3.1) multiplied by 10 9 cfu/g。
The invention also provides a microbial deodorization method of the livestock manure, which comprises the following steps: mixing the solid culture of the Hansenula polymorpha with the livestock and poultry manure to be treated, and stacking.
In the invention, the mass ratio of the solid culture of Hansenula polymorpha to the livestock manure to be treated is preferably 1-2: 50. in the present invention, the thickness of the stack is preferably 30 to 40cm; the stacking time is preferably 48 hours; the stacking preferably comprises 3 to 4 times of turning. The solid culture of the hansenula cerealis is mixed with the livestock manure to be treated and then piled up to promote the fermentation of the hansenula cerealis; the material turning promotes the discharge of gas generated after the fermentation of Hansenula polymorpha.
The following describes the cultivation method of Hansenula polymorpha and the method of deodorizing livestock and poultry manure by microorganism according to the present invention in detail by referring to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The culture method of Hansenula polymorpha comprises the following steps:
(1) Selecting raw materials of sugarcane rind, pineapple rind and ginkgo leaf residue, wherein the water content of the raw materials is 15%, crushing the raw materials into powder with the size of about 0.1cm, weighing 2000 g, 2000 g and 3000g respectively, and the mass ratio is 2:2:3, uniformly mixing, adding 2100mL of water to obtain a solid culture medium, steaming at 100 ℃ and sterilizing for 45min;
(2) Inoculating the inclined plane seed of Hansenula polymorpha into molasses liquid culture medium with 2% sugar concentration, and shaking for 16h at 28 ℃;
(3) Inoculating the cultured liquid seeds into a sterilized solid culture medium, uniformly mixing, controlling the thickness of the culture medium to be 10cm, controlling the ambient temperature to be 28 ℃, culturing for 48 hours, turning the materials once every 12 hours, and spraying a small amount of sterile water on the surface of a material layer to finally obtain a yeast culture;
(4) Calculated by plate viable bacteria, the effective viable bacteria number is 2.2X10 9 CFU/g。
Example 2
The culture method of Hansenula polymorpha comprises the following steps:
(1) Selecting raw materials of sugarcane rind, pineapple rind and ginkgo leaf residue, wherein the moisture content is 15%, crushing the raw materials into powder with the size of about 0.2cm, weighing 4000 g, 4000 g and 5000g respectively, uniformly mixing the raw materials according to the mass ratio of 4:4:5, adding 4550mL of water to ensure that the final moisture content is 50%, obtaining a solid culture medium, steaming the solid culture medium at the temperature of 100 ℃ and sterilizing the solid culture medium for 50min;
(2) Inoculating the inclined plane seed of Hansenula polymorpha into molasses liquid culture medium with 3% sugar concentration, and shaking at 28 ℃ for 18h;
(3) Inoculating the cultured liquid seeds on a sterilized solid culture medium, uniformly mixing, controlling the thickness of the culture medium to be 12cm, controlling the ambient temperature to be 30 ℃, culturing for 54 hours, turning the materials once every 12 hours, and spraying a small amount of sterile water on the surface of a material layer to finally obtain a yeast culture;
(4) Calculated by plate viable bacteria, the effective viable bacteria number is 3.1X10 9 CFU/g。
Example 3
The culture method of Hansenula polymorpha comprises the following steps:
(1) Selecting raw materials of sugarcane rind, pineapple rind and ginkgo leaf residue, wherein the water content is 15%, crushing the raw materials into powder with the size of about 0.4cm, weighing 5000g, 5000g and 6000g respectively, uniformly mixing according to the mass ratio of 5:5:6, adding 6400mL of water to ensure that the final water content is 55%, obtaining a solid culture medium, steaming the solid culture medium at the temperature of 100 ℃ and sterilizing the solid culture medium for 60min;
(2) Inoculating the inclined plane seed of Hansenula polymorpha into molasses liquid culture medium with the sugar concentration of 4%, and shaking for 20 hours at the temperature of 28 ℃;
(3) Inoculating the cultured liquid seeds on a sterilized solid culture medium, uniformly mixing, controlling the thickness of the culture medium to be 15cm, controlling the ambient temperature to be 32 ℃, culturing for 60 hours, turning the materials once every 12 hours, and spraying a small amount of sterile water on the surface of a material layer to finally obtain a yeast culture;
(4) Calculated by plate viable bacteria, the effective viable bacteria number is 2.6X10 9 CFU/g。
Application example 1
1000kg of fresh pig manure is taken, the water content of the pig manure is regulated to be 50 percent, and 80kg of live bacteria with the number of 2.2 multiplied by 10 is weighed 9 CFU/g of Hansenula polymorpha culture of example 1 was added to pig manure, stirred uniformly and then stacked into square stacks of 35cm thickness, with an ambient temperature of 25-30℃being suitable. Turning up and down once every 12 hours; after 20 hours, the odor of the excrement is obviously reduced, and the excrement has a small amount of aromatic flavor; about 40 hours the odor completely disappeared and the fragrance was evident.
Application example 2
The Hansenula polymorpha culture cultured in example 2 is used for deodorizing livestock and poultry manure.
1000kg of fresh chicken manure is taken, and the water content of the chicken manure is adjusted to 60% by adding chaff, wheat bran or crushed straw. Weighing 100 kg of the strain containing 3.1X10 of viable count 9 CFU/g of culture of Hansenula polymorpha is added into chicken manure, and the mixture is uniformly stirred and then piled into square strip piles with the thickness of 30cm, and the environment temperature is 25-30 ℃. Turning up and down once every 12 hours; after 24 hours, the odor of the excrement is obviously reduced, and the excrement has a small amount of aromatic flavor; about 48 hours the odor completely disappeared and the fragrance was evident.
Application example 3
1000kg of garbage doped with feces was taken, the water content thereof was adjusted to 50%, and 50 kg of the viable count of example 3 was weighed to 2.6X10 9 CFU/g culture of Hansenula polymorpha of example 3, adding into garbage, stirring, and stacking into square strip pile with thickness of 40cm, with the temperature of 25-30deg.C being suitable. Turning up and down once every 12 hours; after 18 hours the odor of the waste was significantly reduced, about 36 hours the odor was completely disappeared and a fragrance appeared.
As can be seen from the above examples, the method for culturing Hansenula polymorpha provided by the invention has low equipment requirements; the cost of raw materials is low; the hansenula polymorpha culture obtained by the culture can effectively reduce the release amount of malodorous gases such as ammonia, hydrogen sulfide and the like in the livestock manure; in addition, the odor component is decomposed, and meanwhile, pleasant fragrance is generated, so that the odor masking effect is achieved.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (3)
1. The culture method of Hansenula polymorpha is characterized by comprising the following steps:
(1) Inoculating Hansenula polymorpha in molasses liquid culture medium for culture to obtain liquid culture solution; the culture is shake culture of a shaking table; the temperature of the culture is 28-32 ℃ and the humidity is 70-85%; the culture time is 16-20 h;
(2) Inoculating the liquid culture solution on a solid culture medium for culture to obtain a hansenula polymorpha solid culture; the solid culture medium is prepared from raw materials including sugarcane skin, pineapple skin and ginkgo leaf; the mass ratio of the volume of the liquid culture solution to the solid culture medium is 5-8 mL:100g; the thickness of the solid culture medium is 10-15 cm; the concentration of Hansenula polymorpha in the liquid culture solution is (2-5) multiplied by 10 7 cfu/mL; the temperature of the culture is 28-32 ℃ and the humidity is 70-85%; the culture time is 48-60 hours; the preparation of the solid culture medium comprises the following steps:
(a) Drying sugarcane skin, pineapple skin and ginkgo leaf to obtain dry matters; the mass ratio of the sugarcane rind to the pineapple rind to the ginkgo leaf is 2-4: 2 to 5:3 to 6; the moisture content of the dry matter is less than 15%;
(b) Crushing and mixing the dry matters to obtain a mixture;
(c) Mixing the mixture with water to obtain a solid culture medium; the water content of the solid culture medium is 45-60%;
the effective viable count of the yeast solid culture is 2.2X10 by calculation of plate viable count 9 CFU/g～3.1×10 9 CFU/g。
2. The method for culturing hansenula cerealis according to claim 1, further comprising:
culturing Hansenula polymorpha to obtain a solid Hansenula polymorpha culture;
mixing the solid culture of Hansenula polymorpha with livestock manure to be treated, and stacking; the mass ratio of the solid culture of the Hansenula polymorpha to the livestock manure to be treated is 1-2: 50; the thickness of the pile is 30-40 cm; the stacking time is 48 hours; and turning the materials for 3-4 times during stacking.
3. The method for culturing hansenula polymorpha according to claim 2, wherein the odor eliminating time is 36 to 48 hours after the material turning.
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| CN106916758B (en) * | 2017-05-08 | 2020-03-24 | 中国农业大学 | Hansenula polymorpha and application thereof in wine brewing |
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