CN105316255A - Profitable soil microorganism mixed fermentation method - Google Patents
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Abstract
The invention discloses a profitable soil microorganism mixed fermentation method. The method includes the steps of 1) activating paecilomyces lilacinus, issatchenkia orientalis and bacillus licheniformis on a PDA (potato dextrose agar) medium, a YPD (yeast extract peptone dextrose) medium and a nutrient agar medium respectively, and transferring the three activated strains to corresponding liquid media so as to prepare seed fermentation broth; 2) inoculating 10% of each of the three strains into a mixed fermentation medium, and controlling the living bacteria count of the bacillus licheniformis to be one order of magnitude higher than those of the paecilomyces lilacinus and the issatchenkia orientalis according to the relation between the living bacteria count of the seed fermentation broth and an OD600 (optical density 600) value. The profitable soil microorganism mixed fermentation method has the advantages that a mixed fermentation medium formula is simple, production cost is reduced, equipment utilization rate is increased, and accordingly the profitable soil microorganism mixed fermentation method is suitable for large-scale production of fermented products; the fermented products are capable of improving soil flora balance, promoting growth and increasing production and can be used for producing biofertilizer and biopesticide by being compounded with organic materials, soil improvement agents and the like.
Description
Technical field
The invention belongs to microbial fertilizer technical field, be specifically related to the method for the useful bacterial strain Paecilomyces lilacinus of a kind of soil, Issatchenkia orientalis, Bacillus licheniformis mixed fermentation.
Background technology
Along with the support of intensive agriculture development, policy and people are to the demand of green organic products, while modern agriculture develops rapidly, also encounter many stubborn problems, such as soil compaction, soil-borne disease be serious, degradation under continuous cropping obstacle, crop quality.A large amount of fertilizer and pesticides is manured into soil; have impact on ecotope and human health; and the only way that microniological proudcts address these problems just, especially under the backgrounds such as facing mankind energy dilemma, resource scarcity, environmental pollution (Li Jun, 2006).Microniological proudcts are at culture fertility, improve chemical fertilizer utilization ratio, suppress farm crop to the absorption of nitric nitrogen, heavy metal, agricultural chemicals, purification and rehabilitating soil, reduce corps diseases to occur, and improve the aspects such as crop products quality and food safety and show irreplaceable effect (Pueraria lobota becomes, 2007).
Many important biological processes are can not complete or can only faintly carry out (Lin Jing, 2004) by single microorganism.Complex microorganism preparations contains multiple beneficial microorganism species, effective flora can be bred in plant rhizosphere advantage, improves plant rhizosphere micro-ecological environment, and its meta-bolites and secretory product can stimulate plant growth, improve the receptivity (Li Fudi, 1996) to soil nutrient.Single strain due to its metabolic type, type of respiration and action function single, weak to adaptive capacity to environment, be difficult to become dominant microflora in soil.And mix probiotics due to flora composition various, have adaptive capacity to environment strong, applied range, the advantages such as growth-promoting effect of increasing production is obvious.Therefore, utilize the synergy between microorganism, build the focus that multi-bacteria type microbial preparation is bio-feritlizer industry research.
Soil beneficial microorganism Paecilomyces lilacinus can produce chitinase and serine protease, in the control etc. of nemas, harmful insect, phytopathogen, all have special effect (Li Fang, 2004; Alvarez, 2000).Bakalivanov (1966) finds, Paecilomyces lilacinus can produce indolylacetic acid, has the effect of growth-promoting volume increase.Bacillus licheniformis can promote organism humify in soil, strengthen soil fertility, improve soil compaction, improve soil heat preservation, retaining and accumulation of energy performance, suppress pathogenic bacteria breeding, reduce Plant diseases etc., be widely used in the microbial fertilizer product of Ministry of Agriculture's registration (Hu Yunguang, 2011).(people such as Kurtzman proposed her Sa yeast to be included into pichia spp in 2008 Issatchenkia orientalis, Issatchenkiaorientalis Issatchenkia orientalis, title has changed to Pichiakudriavzevii pichia kudriavzevii) can be used as biological prevention and control agent for antagonism postharvest fruit and vegetable germ (Lin Xiaomin, 2015).Correlative study shows, Paecilomyces lilacinus and Bacillus licheniformis are separately and fertilizer is composite all has disease-resistant growth-promoting effect (Liu Huiqing, 2011).Chinese patent 201010540183.8 discloses a kind of Quick-acting multi-microorganism composite water flush fertilizer and preparation method thereof, is added with the Soil Probiotics such as yeast, Bacillus licheniformis, but its preparation method is each bacterial strain ferment separately after be mixed in proportion.Chinese patent 201410617387.5 discloses a kind of microbial germ powder for rice cultivation and corn, and the beneficial microorganism of interpolation relates to Bacillus licheniformis and yeast etc., but does not illustrate especially for zymotechnique.Chinese patent 200910038426.5 discloses a kind of multifunctional biological compound fertilizer, its preparation method and application, and the beneficial microorganism of interpolation relates to three kinds of genus bacillus, adopts the form of the independent liquid fermenting of tradition.
Summary of the invention
The object of this invention is to provide a kind of Paecilomyces lilacinus, Issatchenkia orientalis, the method for Bacillus licheniformis mixed fermentation and application thereof.
The step of method is:
1) respectively activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis, the three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor.
Wherein, Paecilomyces lilacinus (Paecilomyceslilacinus) is preferably three torches No. 03 bacterial strain, preservation mechanism: China typical culture collection center, preservation place: Wuhan City, Hubei Province. Wuhan University; June 30 2010 preservation time, deposit number CCTCCNO:2010165;
Issatchenkia orientalis (Issatchenkiaorientalis) is three torch-06 bacterial strains preferably, preservation mechanism: China typical culture collection center, preservation place: Wuhan City, Hubei Province. Wuhan University; November 21 2012 preservation time; Deposit number CCTCCNO:2012470
Bacillus licheniformis (Bacilluslincheniformis) is three torch-10 bacterial strains preferably, preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation place, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, April 24 2015 preservation time; Deposit number is CGMCCNo.10741.
Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis preferably adopt PDA, YPD and nutrient agar activation respectively.Wherein:
PDA medium component is: potato 200g/L, glucose 20g/L, agar 20g/L, natural pH; YPD medium component is: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, agar 20g/L, natural pH; Nutrient agar composition is: peptone 10g/L, extractum carnis 3g/L, sodium-chlor 5g/L, agar 20g/L, natural pH.
Paecilomyces lilacinus, after inoculation, is cultivated 4 ~ 6d and is collected spore, prepare spore suspension with brine spore, regulates spore suspension to OD with sterilized water
600be 0.2, spore suspension is inoculated in liquid nutrient medium by the inoculum size with 10%, 28 DEG C, 180rpm cultivates 2d, obtained Paecilomyces lilacinus seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Paecilomyces lilacinus seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=0.3386x-0.0318, R
2=0.9803.
Issatchenkia orientalis, after inoculation, is cultivated 1 ~ 2d from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, is shaken up dilution, record OD
600be 0.2, the inoculum size with 10% is connected to the liquid nutrient medium 28 DEG C of 10mL, 160rpm cultivates 2d, prepare Issatchenkia orientalis seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Issatchenkia orientalis seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=0.6703x-0.0717, R
2=0.9927.
Bacillus licheniformis, after inoculation, is cultivated 2 ~ 3d from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, shakes up dilution, record OD
600be 0.2, the inoculum size with 10% is connected to the liquid nutrient medium 30 DEG C of 10mL, 160rpm cultivates 2d, preparation Bacillus licheniformis seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Bacillus licheniformis seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=2.1695x-0.0659, R
2=0.9976.
2) three strain bacterium are seeded in mixed fermentive culture medium with the inoculum size of 5-20% respectively, according to seed liquor living bacteria count and OD
600relation between value, controls a Bacillus licheniformis order of magnitude higher than the living bacteria count of other two strains bacterium.Preferably, Bacillus licheniformis inoculum size is 0.1-0.5 hundred million/ml fermented liquid, and other two strains bacterium is respectively 0.01-0.05 hundred million/ml fermented liquid.Medium component is: sucrose 10 ~ 20g/L, soyflour 10 ~ 20g/L, potassium primary phosphate 1 ~ 3g/L, trace element 0 ~ 0.5g/L, initial ph value 6.2-6.8.Its medium trace element is calcium chloride or zinc sulfate or does not add.Medium component optimum is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L, initial ph value 6.5.
3) 28 DEG C, 180rpm mixed fermentation 3d.
At present, the method for bibliographical information Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis mixed fermentation is not also had.Nutritive substance needed for the microorganism culturing do not belonged to together is not quite similar, and cultivates simultaneously and easily produces antagonistic action.Except considering carbon source, nitrogenous source, inorganic elements, trace element, water and the oxygen etc. needed for different genera microbial growth.Large scale fermentation is produced, also must pay attention to price and the source of culture medium raw material.Traditional method be different strains is activated in growing environments different separately, collected by centrifugation thalline after fermentation step by step, each tunning is mixed and made into many bacterium mixing prod, has complicated process of preparation, cost is high, and application such as to be restricted at the shortcoming.
Advantage of the present invention is, by three kinds of useful bacterial strain Paecilomyces lilacinus of soil, Issatchenkia orientalis, Bacillus licheniformis mixed fermentation, and fermentation process simple and efficient, in fermenting process, the pH of fermented liquid is more stable; Three kinds of bacterial strains mutually can be worked in coordination with and do not produced antagonistic action again, well-grown in the medium, each function yeast living bacteria count >=200,000,000/mL after fermentation 3d; Mixed fermentive culture medium formula is simple, reduces production cost, improves plant factor, be suitable for the large-scale production of leavened prod; Mixed fermenting agent has and improves soil colony balance, reduces the effect that fertilizer and pesticide is used; Mixed fermenting agent use safety, environmentally safe.
The mixed fungus fermentation method of three kinds of bacterium; on the one hand for the large-scale production of leavened prod provides method; on the other hand; leavened prod can with organic materials (feces of livestock and poultry become thoroughly decomposed, natural pond slag, bagasse, peat, stalk etc.) composite production biological organic fertilizer and and the composite production biological compound fertilizer such as trace element, soil improvement agent, while also can as biological pesticide.Mixed fungus fermentation method of the present invention is that the production application of product lays the foundation.
Accompanying drawing explanation
The colonial morphology of Fig. 1 tri-strain bacterium on basic medium.
Fig. 2 different carbon source is on the impact of three strain bacterium living bacteria counts.
Fig. 3 different nitrogen sources is on the impact of three strain bacterium living bacteria counts.
The different inorganic salt of Fig. 4 are on the impact of three strain bacterium living bacteria counts.
The impact of the different trace elements of Fig. 5 on three strain bacterium living bacteria counts.
Fig. 6 tri-strain bacterium is mutual not antagonism when mixed culture.
Embodiment
Example will further illustrate feasibility and the effect of the inventive method below.
Embodiment 1 mixed culture medium formulation optimization
Substratum based on extractum carnis 3g/L, peptone 12g/L, glucose 20g/L, potassium primary phosphate 2g/L, zinc sulfate 0.1g/L, culture condition is initial ph value 6.5,28 DEG C, 180rpm vibrates 3d.Can observe three strain bacterium by microscopy and culture medium flat plate coating, based on Fig. 1, the colonial morphology of three strain bacterium on substratum, shows that this basic medium is applicable to the cultivation of three strain bacterium.
The glucose in basic medium is substituted respectively with the addition sucrose of 20g/L, maltose, Zulkovsky starch, investigate their impacts on the living bacteria count of three strain bacterium of mixed culture, as shown in Figure 2, when taking Zulkovsky starch as carbon source, total living bacteria count of three strain bacterium is the highest, is thereafter sucrose, maltose, glucose successively.Zulkovsky starch and sucrose, maltose on living bacteria count to affect difference remarkable, considering cost, selection sucrose is carbon source.
Extractum carnis in basic medium and peptone is substituted respectively with the addition wheat bran of 15g/L, soyflour, yeast powder, investigate their impacts on the living bacteria count of three strain bacterium of mixed culture, as shown in Figure 3, during with extractum carnis and peptone for nitrogenous source, total living bacteria count of three strain bacterium is the highest, is thereafter soyflour, yeast powder, wheat bran successively.Extractum carnis and peptone and soyflour on living bacteria count to affect difference remarkable, considering cost, selection soyflour is nitrogenous source.
The potassium primary phosphate in basic medium is substituted respectively with the addition sodium-chlor of 1g/L, magnesium sulfate, investigate their impacts on the living bacteria count of three strain bacterium of mixed culture, as shown in Figure 4, when taking potassium primary phosphate as inorganic salt, total living bacteria count of three strain bacterium is the highest, is thereafter magnesium sulfate, sodium-chlor successively.Therefore select potassium primary phosphate as the inorganic salt source in substratum.
The zinc sulfate in basic medium is substituted respectively with the addition copper sulfate of 0.1g/L, ferrous sulfate, calcium chloride, investigate their impacts on the living bacteria count of three strain bacterium of mixed culture, as shown in Figure 5, when taking zinc sulfate as trace element, total living bacteria count of three strain bacterium is the highest, is thereafter calcium chloride, copper sulfate, ferrous sulfate successively.Zinc sulfate and calcium chloride on living bacteria count to affect difference remarkable, considering cost, selective chlorination calcium is as the trace element source in substratum.
Select optimum carbon source, nitrogenous source, inorganic salt and trace element by L respectively
9(3)
4design four factor three horizontal quadrature tests, 9 combinations, repeat 3 times.
Table 1 medium optimization orthogonal test factor and level (unit: g/L)
Test-results is known, and the extreme difference of soyflour is maximum, is the important factor affecting three strain bacterium living bacteria counts, and be secondly potassium primary phosphate, sucrose successively, be finally calcium chloride, investigate each factor level value by living bacteria count, optimal medium is combined as A
2b
3c
3d
3, i.e. sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L.
Table 2 medium optimization orthogonal experimental design and result
The culture effect of embodiment 2 bacterial strain defined medium and mixed fermentive culture medium compares
Issatchenkia orientalis with from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, shakes up and is diluted to OD after overactivation
600be 0.2, the inoculum size with 10% be inoculated in respectively following three kinds known cultivate Issatchenkia orientalis liquid nutrient medium and mixed fermentation liquid nutrient medium of the present invention in, 28 DEG C, 180rpm cultivates 48h, sampling detects living bacteria count.
The substratum of table 3 three kinds of known Issatchenkia orientalis and mixed fermentive culture medium of the present invention formula
Bacillus licheniformis from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, shakes up and is diluted to OD after overactivation
600be 0.2, the inoculum size with 10% be inoculated in following three kinds known cultivate Bacillus licheniformis liquid nutrient medium and mixed fermentation liquid nutrient medium of the present invention in, 28 DEG C, 180rpm cultivates 48h, sampling detects living bacteria count.
The substratum of table 4 three kinds of known Bacillus licheniformis and mixed fermentive culture medium of the present invention formula
The dull and stereotyped 5d of Paecilomyces lilacinus coated plate collects spore, prepares spore suspension, shake up and be diluted to OD with brine spore
600be 0.2, the inoculum size with 10% spore suspension is inoculated in following three kinds known cultivate Paecilomyces lilacinus liquid nutrient medium and mixed fermentation liquid nutrient medium of the present invention in, 28 DEG C, 180rpm cultivate 72h sampling detect living bacteria count.
The substratum of table 5 three kinds of known Paecilomyces lilacinus and mixed fermentive culture medium of the present invention formula
Under the prerequisite controlling identical inoculum size, after Issatchenkia orientalis cultivates 24h in A, B, C, D tetra-kinds of different culture medias, living bacteria count is respectively 2.6,0.6,4,1.9 hundred million/mL, after Bacillus licheniformis cultivates 48h in A, B, C, D tetra-kinds difference is cultivated, living bacteria count is respectively 0.89,0.02,0.25,1.2 hundred million/mL, after Paecilomyces lilacinus cultivates 72h in A, B, C, D tetra-kinds difference is cultivated, living bacteria count is respectively 0.2,0.15,0.25,8.3 hundred million/mL.Result shows, and three kinds of microorganisms all well can grow on mixed fermentive culture medium of the present invention, and substratum of the present invention is also applicable to the demand only need producing a kind of bacterium.Consider price and the source of culture medium raw material, mixed fermentive culture medium formula of the present invention is that the large-scale application of three bacterium provides necessary precondition.
The Quality Control of embodiment 3 mixed fermentation inoculum size
In order to control the initial inoculation living bacteria count of the various bacterium of mixed fermentation system, make the initial living bacteria count of various bacterium in known controlled range, in conjunction with seed liquor living bacteria count and OD
600relation between value, has made three strain bacterium seed liquor living bacteria count and OD
600typical curve between value.
Paecilomyces lilacinus, after inoculation, is cultivated 4 ~ 6d and is collected spore, prepare spore suspension with brine spore, regulates spore suspension to OD with sterilized water
600be 0.2, spore suspension is inoculated in liquid nutrient medium by the inoculum size with 10%, 28 DEG C, 180rpm cultivates 2d, obtained Paecilomyces lilacinus seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Paecilomyces lilacinus seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=0.3386x-0.0318, R
2=0.9803.
Issatchenkia orientalis, after inoculation, is cultivated 1 ~ 2d from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, is shaken up dilution, record OD
600be 0.2, the inoculum size with 10% is connected to the liquid nutrient medium 28 DEG C of 10mL, 160rpm cultivates 2d, prepare Issatchenkia orientalis seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Issatchenkia orientalis seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=0.6703x-0.0717, R
2=0.9927.
Bacillus licheniformis, after inoculation, is cultivated 2 ~ 3d from inclined-plane scraping one ring thalline in the triangular flask that physiological saline is housed, shakes up dilution, record OD
600be 0.2, the inoculum size with 10% is connected to the liquid nutrient medium 30 DEG C of 10mL, 160rpm cultivates 2d, preparation Bacillus licheniformis seed liquor, through dilution coated plate counting and OD
600the measurement of value, draws Bacillus licheniformis seed liquor living bacteria count (hundred million/mL) and OD
600pass between value is: y=2.1695x-0.0659, R
2=0.9976.
Three strain bacterium seed liquor are seeded in mixed fermentive culture medium, according to seed liquor living bacteria count and OD with the inoculum size of 10% respectively
600relation between value, reasonably dilutes seed liquor, controls a Bacillus licheniformis order of magnitude higher than the living bacteria count of other two strains bacterium, can significantly improve the living bacteria count of mixed fungus fermentation terminal.Control the ratio of each bacterial strain living bacteria count during mixed fungus fermentation, be conducive to working in coordination with mutually between each bacterium, reach optimum ferment effect.
Embodiment 4 three strain bacterium is mutual not antagonism when mixed culture
The three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor by activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis on PDA, YPD, nutrient agar respectively.
By scribe point connection, the sterilizing toothpick speckling with Issatchenkia orientalis and Bacillus licheniformis seed liquor is intersected and lines on mixed fermentive culture medium flat board of the present invention, connect the Paecilomyces lilacinus seed liquor of 100 μ L at region intermediate point, as shown in Figure 6 simultaneously.Medium component is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L, agar 20g/L, initial ph value 6.5.
Cultivate 2 ~ 3d, observe the interaction between three kinds of bacterial strains for 28 DEG C.
As shown in Figure 6, soil beneficial microorganism Paecilomyces lilacinus, Bacillus licheniformis, Issatchenkia orientalis well can grow in the mixed fermentive culture medium of invention, mutually not antagonism.Utilizing mixed culture to produce three bacterium leavened prods is feasible and optimism.
The comparison that embodiment 5 mixed-strains liquid fermentation and each bacterial strain ferment separately
1) the three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor by activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis on PDA, YPD, nutrient agar respectively.
2) in different shaking flasks, access Issatchenkia orientalis, Bacillus licheniformis, Paecilomyces lilacinus respectively, and in same shaking flask, access the seed liquor of three strain bacterium simultaneously, at 28 DEG C, cultivate in the shaking table of 180rpm.The seed liquor inoculum size of often kind of bacterium is all 10%, according to seed liquor living bacteria count and OD in mixed fermentation shaking flask
600relation between value, controls a Bacillus licheniformis order of magnitude higher than the living bacteria count of other two strains bacterium.Cultivate 3d, detect pH, living bacteria count every 24h sampling.Medium component is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L, initial ph value 6.5.
3) wherein Issatchenkia orientalis YPD culture medium flat plate counts, and Bacillus licheniformis nutrient agar medium counts, and Paecilomyces lilacinus rose bengal medium counts.
Measurement result is as shown in table 6, and mixing fermentation culture is compared with fermenting separately, and Bacillus licheniformis living bacteria count is increased to 5,200,000,000/mL when 72h, illustrates that the growth of the tunning of three bacterium to Bacillus licheniformis has promoter action; The growth of Paecilomyces lilacinus presents the trend increased progressively, and when 72h, living bacteria count reaches 3.2 hundred million/mL.And the pH value amplitude of fluctuation of fermented liquid is little, illustrate that three bacterium mixed fermentations are more stable.
The comparison of table 6 soil beneficial microorganism mixed fungus fermentation and separately fermentation living bacteria count
Note: Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis are respectively referred to as pale purple, lichens, east.
The impact of the different trace elements of embodiment 6 on mixed fermentation living bacteria count
1) the three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor by activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis on PDA, YPD, nutrient agar respectively.
2) mixed fermentive culture medium composition is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, micro-0.1g/L, initial ph value 6.5.Adding trace element for being formula 1 during calcium chloride, adding trace element for being formula 2 during zinc sulfate, not adding trace element is formula 3.In three kinds of culture medium prescription shaking flasks, access three strain bacterium, the seed liquor inoculum size of often kind of bacterium is all 10%, does not do the Quality Control of seed liquor living bacteria count, 28 DEG C, cultivates 3d in the shaking table of 180rpm, and terminal sampling detects pH, living bacteria count.
3) wherein Issatchenkia orientalis YPD culture medium flat plate counts, and Bacillus licheniformis nutrient agar medium counts, and Paecilomyces lilacinus rose bengal medium counts.
Measurement result is as shown in table 7, and in 3 kinds of shaking flasks containing different trace element, three strain bacterium are energy normal growth all, each living bacteria count >=200,000,000/mL during fermentation termination.
The comparison of three bacterium living bacteria counts and pH in the different trace element formula of table 7
Note: Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis are respectively referred to as pale purple, lichens, east.
Embodiment 7 soil beneficial microorganism tunning is to the growth-promoting production-increasing function of Lettuce
1) the three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor by activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis on PDA, YPD, nutrient agar respectively.
2) Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis, the seed liquor living bacteria count of three strain bacterium is according to the typical curve between living bacteria count and OD, control at a Bacillus licheniformis order of magnitude higher than the living bacteria count of other two bacterial strains, be seeded in mixed fermentive culture medium with the inoculum size of 10% respectively, medium component is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L, initial ph value 6.5.
3) 28 DEG C, 180rpm mixed fermentation 3d, collect tunning.
4) test plant is Lettuce, and select the consistent seedling of growing way to carry out pot experiment, 4 process are established in test, repeat 5 times, the strain of every basin 5 ~ 6, and test Treatment Design is as table 8.
Table 8 Lettuce pot experiment Treatment Design
5) each process be placed in there is taking shelter from rain, ventilate, the greenhouse of the function of temperature adjustment, unifiedly carry out daily spraying and water, respectively at measuring the long and fresh amount of plant height, root after 10d, 20d, 30d, 40d.Measurement result is analyzed as table 9.The process 1 of using zymocyte liquid, compared with other control treatment, can significantly improve the biomass of Lettuce in each sampling period.And process 2 (conventional fertilizer application+inactivated bacterial liquid) and process 3 (conventional fertilizer applications), front 30d sampling, between two process, fresh weight difference is not remarkable, 40d, and the biomass of process 2 is significantly higher than process 3.
Under table 9 different treatment, Lettuce biomass over time
Claims (9)
1. a method for soil beneficial microorganism mixed fermentation, is characterized in that, method steps is:
1) the three strain bacterium activated are transferred on corresponding liquid nutrient medium and prepare seed liquor by difference activated strains Paecilomyces lilacinus, Issatchenkia orientalis, Bacillus licheniformis;
2) three strain bacterium are seeded in mixed fermentive culture medium with the inoculum size of 5-20% volume ratio respectively, mixed fermentive culture medium composition comprises sucrose 10 ~ 20g/L, soyflour 10 ~ 20g/L, potassium primary phosphate 1 ~ 3g/L, trace element 0 ~ 0.5g/L, initial ph value 6.2-6.8;
3) mixed fermentation.
2. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, it is characterized in that: described step 1) in, described Paecilomyces lilacinus (Paecilomyceslilacinus) is three torches No. 03 bacterial strain, deposit number is CCTCCNO:2010165, described Issatchenkia orientalis (Issatchenkiaorientalis) is three torch-06 bacterial strains, deposit number is CCTCCNO:2012470, and described Bacillus licheniformis (Baclicuslincheniformis) is three torch-10 bacterial strains, deposit number is CGMCCNo.10741.
3. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, is characterized in that: described step 2) in, according to seed liquor living bacteria count and OD
600relation between value, controls a Bacillus licheniformis order of magnitude higher than the living bacteria count of other two strains bacterium.
4. the method for a kind of soil beneficial microorganism according to claim 3 mixed fermentation, is characterized in that: Bacillus licheniformis inoculum size is 0.1-0.5 hundred million/ml, and other two strains bacterium is respectively 0.01-0.05 hundred million/ml.
5. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, is characterized in that: described step 2) in, the trace element in mixed fermentive culture medium composition is calcium chloride or zinc sulfate or does not add.
6. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, it is characterized in that: described step 2) in, mixed fermentive culture medium composition is: sucrose 10 ~ 20g/L, soyflour 10 ~ 20g/L, potassium primary phosphate 1 ~ 3g/L, trace element 0.1 ~ 0.5g/L, initial ph value 6.5, described trace element is calcium chloride.
7. the method for a kind of soil beneficial microorganism according to claim 5 mixed fermentation, is characterized in that: mixed fermentive culture medium is: sucrose 15g/L, soyflour 10g/L, potassium primary phosphate 1g/L, calcium chloride 0.1g/L, initial ph value 6.5.
8. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, is characterized in that: described step 3) in, 20-30 DEG C, 150-250rpm mixed fermentation 2 ~ 3d.
9. the method for a kind of soil beneficial microorganism according to claim 1 mixed fermentation, is preparing the application in bio-fertilizer and biological pesticide.
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| CN201510390509.6A Active CN105316255B (en) | 2015-07-06 | 2015-07-06 | A kind of method of soil beneficial microbe mixed fermentation |
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| CN107573941A (en) * | 2017-08-30 | 2018-01-12 | 覃祖桓 | Application and its method of a kind of storehouse Delhi A Ziweishi Pichia yeasts in soil |
| CN110818508A (en) * | 2019-12-04 | 2020-02-21 | 河南农贝得农业科技有限公司 | Microbial agent with function of breaking soil hardening and preparation method thereof |
| CN111001656A (en) * | 2019-12-26 | 2020-04-14 | 湖南素里养农业科技有限公司 | Soil cultivation method for reducing pesticide residue |
| CN112225595A (en) * | 2020-10-21 | 2021-01-15 | 福建三炬生物科技股份有限公司 | Water-soluble compound microbial fertilizer and preparation method thereof |
| CN114907991A (en) * | 2022-05-09 | 2022-08-16 | 漳州三炬生物技术有限公司 | Compound microorganism for decomposing banana straws, banana straw decomposing agent, banana straw organic fertilizer and application |
| CN115232762A (en) * | 2022-06-07 | 2022-10-25 | 南京农业大学 | Mixed fermentation method of composite flora |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107573941A (en) * | 2017-08-30 | 2018-01-12 | 覃祖桓 | Application and its method of a kind of storehouse Delhi A Ziweishi Pichia yeasts in soil |
| CN110818508A (en) * | 2019-12-04 | 2020-02-21 | 河南农贝得农业科技有限公司 | Microbial agent with function of breaking soil hardening and preparation method thereof |
| CN111001656A (en) * | 2019-12-26 | 2020-04-14 | 湖南素里养农业科技有限公司 | Soil cultivation method for reducing pesticide residue |
| CN112225595A (en) * | 2020-10-21 | 2021-01-15 | 福建三炬生物科技股份有限公司 | Water-soluble compound microbial fertilizer and preparation method thereof |
| CN114907991A (en) * | 2022-05-09 | 2022-08-16 | 漳州三炬生物技术有限公司 | Compound microorganism for decomposing banana straws, banana straw decomposing agent, banana straw organic fertilizer and application |
| CN114907991B (en) * | 2022-05-09 | 2024-03-29 | 漳州三炬生物技术有限公司 | Composite microorganism for decomposing banana straw, banana straw decomposing agent, banana straw organic fertilizer and application |
| CN115232762A (en) * | 2022-06-07 | 2022-10-25 | 南京农业大学 | Mixed fermentation method of composite flora |
| CN115232762B (en) * | 2022-06-07 | 2023-10-10 | 南京农业大学 | Mixed fermentation method of composite flora |
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| CN105316255B (en) | 2018-08-14 |
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