CN100344751C - Method for batch-culturing bacillus subtilis variant strain by using solid fermentation - Google Patents
Method for batch-culturing bacillus subtilis variant strain by using solid fermentation Download PDFInfo
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Abstract
The present invention relates to a method for culturing bacillus subtilis variant strains in batch by solid fermentation, which belongs to the technical field of biologic material culture. The operation comprises the following steps: the preparation of a solid culture medium, sterilization, inoculated culture and viable bacterium harvest. The present invention has the advantages that wet bacteria do not need to be collected by a special centrifugal machine, namely that the device investment is low, a large quantity of viable bacteria are collected, and the present invention has no problems of waste liquid discharge or environmental pollution. The method is suitable for culturing wet bacteria of bacillus subtilis variant strains in batch, provides raw materials of viable bacteria for the industrialized production of composite water purifiers of biological viable bacteria and is a new technology capable of promoting aquaculture.
Description
Technical field
The present invention relates to the method for a kind of batch-culturing bacillus subtilis variant strain by using solid fermentation (Bucillus Subtilis sp.), definitely say, relate to a kind of method, belong to biomaterial culture technique field by solid fermentation process batch-culturing bacillus subtilis variant strain by using.
Background technology
The Chi Shui of aquaculture pond mainly is subjected to two kinds of pollutions: a large amount of human lives's sewage, enter river, lake as people's domestic animals manure, washing water etc., make and contain a large amount of ammonia nitrogens and nitrate and organism in river, the lake water, above-mentioned Chi Shui Ji Zihe, lake, this Chi Shuizhong also contains a large amount of ammonia nitrogens and nitrate and organism; Above-mentioned Chi Shui also is subjected to feeding the remaining feed and the excremental pollution of fishery products of fishery products, and the contained macromolecular organic compound of pollutent causes the eutrophication of water body and poisonous algae and bacterium to breed in a large number.Above-mentioned two kinds of pollutions cause fishery products, as fish, shrimp, the sick death of crab.
As everyone knows, in natural soil, hay etc., a class subtilis is arranged.This genera bacillus can be decomposed remaining feed and the excremental larger molecular organics of aquatic products, can transform ammonia nitrogen and nitrate in the water of aquaculture pond pond again.Especially the bacillus subtilis variant strain by using that optimizes from this bacillus more has vitality strong, breeds fast advantage.Above-mentioned dissociant obviously is the bacterial classification source that ideal prepares compound biological water purifiers with live bacteria.The inventor application number and denomination of invention be respectively 01112787.2 and the application for a patent for invention of " a kind of batch culture process of bacillus subtilis variant strain by using " in the method for cultivating above-mentioned dissociant in batches by the liquid fermenting process is proposed, utilize this method can prepare a large amount of above-mentioned dissociants, be the wet thallus of bacillus subtilis variant strain by using, lay a good foundation for producing compound biological water purifiers with live bacteria in batches.But this method still has following shortcoming: need be with special supercentrifuge when gathering in the crops above-mentioned wet thallus, and facility investment is big; In the waste liquid behind the results wet thallus, still have a large amount of viable bacterias not to be separated, i.e. wet thallus yield loss is bigger, causes production cost to rise; Liquid waste disposal difficulty behind the results wet thallus enters environment and can cause new pollution.
Summary of the invention
The technical problem to be solved in the present invention is to release a kind of method by solid fermentation process batch-culturing bacillus subtilis variant strain by using.When utilizing this method to cultivate above-mentioned dissociant, when the wet thallus of the above-mentioned dissociant of results, do not need supercentrifuge; Wet thallus yield height; Whole fermentation process does not produce waste liquid, does not have discharging of waste liquid and environmental pollution problems.
The present invention is by solving the problems of the technologies described above by the following technical solutions: a kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that operation steps:
The 1st step preparation solid medium, weight percent prescription by this substratum, soybean cake powder 2～60%, Semen Maydis powder 2～60%, rice bran 2～40%, wheat bran 2～40%, ammonium sulfate 0.4% and ammonium chloride 0.2%, take by weighing solid material, ammonium sulfate and ammonium chloride are dissolved in 30～40% the water that weight is the solid material gross weight, mixed with all the other solid materials again, stir, be divided into four parts, drop into four solid-state fermentation tanks respectively, make four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05～1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, 121～125 ℃ and 6～60rph condition under, four jars of solid mediums are carried out autoclaving handle, the treatment time is 30～60 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40～50 ℃, under the situation of fermentor tank rotation, to every jar spray into weight be every jar in 30～40% sterilized water of solid material weight, when jar temperature drop to 30～40 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, every jar inoculum size is 2～3% of every jar of solid material weight, is respectively 0.2～0.8kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600～2000L/h, 6～60rph and 30～40 ℃ condition under, four jars of dissociants are continuously fermented cultivated 20～50 hours;
The 4th step was gathered in the crops viable bacteria, handled through the last step, above-mentioned dissociant is bred in a large number, gather in the crops the wet thallus of a large amount of bacillus subtilis variant strain by using respectively from four fermentor tanks, the weight of every kind of wet thallus with drop into every jar in being equal in weight of solid material and water, every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
Compare with background technology, the present invention has following outstanding effect:
The weight of the wet thallus of every jar of results roughly with drop into every jar in being equal in weight of solid material and water, not only wet thallus yield height, and wet thallus does not need special whizzer just can collect, and helps to reduce significantly facility investment.
2. Shou Huo viable count is many, as adopting liquid culture method to cultivate with 500L liquid culture jar, only can obtain wet thallus 30～35kg, as use method of the present invention instead and cultivate, the results wet thallus is in 210kg, and that is not cultivated with method of the present invention, and the wet thallus of results is 6～7 times of liquid culture method approximately;
3. the fermentation culture process does not produce waste liquid, no discharging of waste liquid and problem of environmental pollution.
Embodiment
Embodiment 1.A kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 120kg, wherein soybean cake powder 36kg, Semen Maydis powder 36kg, rice bran 24kg, wheat bran 23.28kg, ammonium sulfate 0.48kg and ammonium chloride 0.24kg, ammonium sulfate and ammonium chloride are dissolved in the water of 42kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 60rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 55 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 42kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 3kg, is respectively 0.7kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 2000L/h, 60rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 48 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 210kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
Embodiment 2.A kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 60kg, wherein soybean cake powder 3kg, Semen Maydis powder 33kg, rice bran 20.64kg, wheat bran 3kg, ammonium sulfate 0.24kg and ammonium chloride 0.12kg, ammonium sulfate and ammonium chloride are dissolved in the water of 19.2kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 121 ℃ and the 6rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 35 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 19.2kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 1.2kg, is respectively 0.3kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600L/h, 6rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 24 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 100kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
Embodiment 3.A kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 90kg, wherein soybean cake powder 4.5kg, Semen Maydis powder 49.5kg, rice bran 4.5kg, wheat bran 30.96kg, ammonium sulfate 0.36kg and ammonium chloride 0.18kg, ammonium sulfate and ammonium chloride are dissolved in the water of 28.8kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 121 ℃ and the 30rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 40 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 28.8kg, when jar temperature drop to 37 ℃, to insert last described four fermentor tanks of two steps respectively through the bacterium seed of two bacillus subtilis variant strain by using of cultivating that are in logarithmic phase in advance, every jar inoculum size is 2.7kg, is respectively 0.5kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1800L/h, 30rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 36 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 150kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
Embodiment 4.A kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 60kg, wherein soybean cake powder 33kg, Semen Maydis powder 3kg, rice bran 20.64kg, wheat bran 3kg, ammonium sulfate 0.24kg and ammonium chloride 0.12kg, ammonium sulfate and ammonium chloride are dissolved in the water of 22.8kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 6rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 35 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 22.8kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 1.2kg, is respectively 0.3kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600L/h, 6rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 24 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 107kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
Embodiment 5.A kind of method of batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 90kg, wherein soybean cake powder 49.5kg, Semen Maydis powder 4.5kg, rice bran 4.5kg, wheat bran 30.96kg, ammonium sulfate 0.36kg and ammonium chloride 0.18kg, ammonium sulfate and ammonium chloride are dissolved in the water of 34.2kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 30rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 40 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 34.2kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 2.7kg, is respectively 0.5kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1800L/h, 30rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 36 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 161kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
The present invention is particularly suitable for being used for the wet thallus of batch-culturing bacillus subtilis variant strain by using, for the compound viable bacteria water purification agent of suitability for industrialized production provides the viable bacteria raw material, be one can promote aquaculture, can produce new technology great economic benefit and that be convenient to promote the use of.
Claims (6)
1. the method for a batch-culturing bacillus subtilis variant strain by using solid fermentation is characterized in that, operation steps:
The 1st step preparation solid medium, weight percent prescription by this substratum, soybean cake powder 2～60%, Semen Maydis powder 2～60%, rice bran 2～40%, wheat bran 2～40%, ammonium sulfate 0.4% and ammonium chloride 0.2%, take by weighing solid material, ammonium sulfate and ammonium chloride are dissolved in 30～40% the water that weight is the solid material gross weight, mixed with all the other solid materials again, stir, be divided into four parts, drop into four solid-state fermentation tanks respectively, make four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05～1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, 121～125 ℃ and 6～60rph condition under, four jars of solid mediums are carried out autoclaving handle, the treatment time is 30～60 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40～50 ℃, under the situation of fermentor tank rotation, to every jar spray into weight be every jar in 30～40% sterilized water of solid material weight, when jar temperature drop to 30～40 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, every jar inoculum size is 2～3% of every jar of solid material weight, is respectively 0.2～0.8kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600～2000L/h, 6～60rph and 30～40 ℃ condition under, four jars of dissociants are continuously fermented cultivated 20～50 hours;
The 4th step was gathered in the crops viable bacteria, handled through the last step, above-mentioned dissociant is bred in a large number, gather in the crops the wet thallus of a large amount of bacillus subtilis variant strain by using respectively from four fermentor tanks, the weight of every kind of wet thallus with drop into every jar in being equal in weight of solid material and water, every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
2. the method for batch-culturing bacillus subtilis variant strain by using solid fermentation according to claim 1 is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 120kg, wherein soybean cake powder 36kg, Semen Maydis powder 36kg, rice bran 24kg, wheat bran 23.28kg, ammonium sulfate 0.48kg and ammonium chloride 0.24kg, ammonium sulfate and ammonium chloride are dissolved in the water of 42kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 60rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 55 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 42kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 3kg, is respectively 0.7kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 2000L/h, 60rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 48 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 210kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
3. the method for batch-culturing bacillus subtilis variant strain by using solid fermentation according to claim 1 is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 60kg, wherein soybean cake powder 3kg, Semen Maydis powder 33kg, rice bran 20.64kg, wheat bran 3kg, ammonium sulfate 0.24kg and ammonium chloride 0.12kg, ammonium sulfate and ammonium chloride are dissolved in the water of 19.2kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 121 ℃ and the 6rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 35 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 19.2kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 1.2kg, is respectively 0.3kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600L/h, 6rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 24 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 100kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
4. the method for batch-culturing bacillus subtilis variant strain by using solid fermentation according to claim 1 is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 90kg, wherein soybean cake powder 4.5kg, Semen Maydis powder 49.5kg, rice bran 4.5kg, wheat bran 30.96kg, ammonium sulfate 0.36kg and ammonium chloride 0.18kg, ammonium sulfate and ammonium chloride are dissolved in the water of 28.8kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.05kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 121 ℃ and the 30rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 40 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 28.8kg, when jar temperature drop to 37 ℃, to insert last described four fermentor tanks of two steps respectively through the bacterium seed of two bacillus subtilis variant strain by using of cultivating that are in logarithmic phase in advance, every jar inoculum size is 2.7kg, is respectively 0.5kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1800L/h, 30rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 36 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 150kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
5. the method for batch-culturing bacillus subtilis variant strain by using solid fermentation according to claim 1 is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 60kg, wherein soybean cake powder 33kg, Semen Maydis powder 3kg, rice bran 20.64kg, wheat bran 3kg, ammonium sulfate 0.24kg and ammonium chloride 0.12kg, ammonium sulfate and ammonium chloride are dissolved in the water of 22.8kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 6rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 35 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 22.8kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 1.2kg, is respectively 0.3kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1600L/h, 6rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 24 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 107kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
6. the method for batch-culturing bacillus subtilis variant strain by using solid fermentation according to claim 1 is characterized in that, operation steps:
The 1st step preparation solid medium, it is the solid-state fermentation tank preparation solid medium that four capacity are 500L, dropping into weight to every jar is the solid material of 90kg, wherein soybean cake powder 49.5kg, Semen Maydis powder 4.5kg, rice bran 4.5kg, wheat bran 30.96kg, ammonium sulfate 0.36kg and ammonium chloride 0.18kg, ammonium sulfate and ammonium chloride are dissolved in the water of 34.2kg, inject every jar, stir, be mixed with four jars of identical solid mediums;
The sterilization of the 2nd step is respectively 1.3kg/cm at pressure, temperature and fermentor tank rotating speed
2, under 125 ℃ and the 30rph condition, four jars of solid mediums are carried out autoclaving handle, the treatment time is 40 minutes;
The 3rd step inoculation culture, during jar temperature drop to 40 ℃, under the situation of fermentor tank rotation, to every jar of sterilized water that sprays into 34.2kg, when jar temperature drop to 37 ℃, the bacterium seed of the bacillus subtilis variant strain by using that is in logarithmic phase cultivated through secondary is in advance inserted last described four fermentor tanks of two steps respectively, and every jar inoculum size is 2.7kg, is respectively 0.5kg/cm in tank pressure, ventilation, fermentor tank rotating speed and a jar temperature
2, 1800L/h, 30rph and 37 ℃ condition under, four jars of dissociants are continuously fermented cultivated 36 hours;
The 4th step results viable bacteria, after the last step handled, above-mentioned dissociant was bred in a large number, gathered in the crops the wet thallus 161kg of bacillus subtilis variant strain by using respectively from four fermentor tanks, and every gram wet thallus contains viable count 2,000,000,000～5,000,000,000.
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| CN101333499B (en) * | 2008-05-13 | 2010-06-09 | 昆山工研院华科生物高分子材料研究所有限公司 | Complex active bacterial biological water purifying a gent and method for preparing same |
| CN102352334B (en) * | 2011-10-21 | 2013-01-09 | 山东省农业科学院高新技术研究中心 | Method for producing viable bacillus subtilis by way of solid fermentation |
| CN103881953B (en) * | 2014-04-18 | 2016-08-10 | 哈尔滨方依达肥业股份有限公司 | A kind of continuous ferment process of bacillus subtilis |
| CN104946565A (en) * | 2015-06-29 | 2015-09-30 | 浙江惠嘉生物科技有限公司 | Solid-state continuous fermentation method of Bacillus subtilis |
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| CN1113951A (en) * | 1994-05-24 | 1995-12-27 | 中国科学院遗传研究所 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1113951A (en) * | 1994-05-24 | 1995-12-27 | 中国科学院遗传研究所 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
| CN1177637A (en) * | 1997-09-26 | 1998-04-01 | 北京英特莱幸诺生物技术有限责任公司 | Preparation of living bacillus subtilis and preparation method thereof |
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