CN109679876A

CN109679876A - Complex microorganism symbiosis Fiber differentiation technology and microbe symbiotic culture microbial inoculum and its application based on SOD

Info

Publication number: CN109679876A
Application number: CN201910046027.7A
Authority: CN
Inventors: 王代军
Original assignee: Zhejiang Daijun Biomedicine Science & Technology Co Ltd
Current assignee: Zhejiang Daijun Biomedicine Science & Technology Co Ltd
Priority date: 2019-01-17
Filing date: 2019-01-17
Publication date: 2019-04-26

Abstract

The present invention relates to microbe symbiotic culture fields, specifically, providing a kind of complex microorganism symbiosis Fiber differentiation technology based on SOD and microbe symbiotic culture microbial inoculum and its application.The abductive approach of microbe symbiotic culture provided by the invention, demand according to aimed strain to oxygen content is classified, the close bacterial strain of oxygen demand is first merged into culture and obtains several bacterial strain groups, the induction of symbiosis culture since the minimum bacterial strain group of oxygen demand, the adaptability of system is promoted by the amount of bioactivity superoxide dismutase in guarantee system and the oxygen content being gradually increased in condition of culture, when oxygen content reaches a certain concentration, bacterial strain group similar in oxygen content requirements is added, system is promoted to the adaptability of more oxygen content by the amount of superoxide dismutase in guarantee system and the oxygen content being gradually increased in condition of culture again, repeat above-mentioned operation, until all bacterial strain group symbiosis cultures are completed, obtain microbe symbiotic culture microbial inoculum.

Description

Complex microorganism symbiosis Fiber differentiation technology and microbe symbiotic culture based on SOD Microbial inoculum and its application

Technical field

The present invention relates to microbe symbiotic culture fields, in particular to a kind of complex microorganism symbiosis based on SOD Fiber differentiation technology and microbe symbiotic culture microbial inoculum and its application.

Background technique

People experienced natural mixed culture, pure culture to purposive mixed culture (co-cultivation) to the utilization of microorganism Stage.In recent decades, people gradually have found have some biochemical processes that two or more microorganisms is needed just to can be carried out, and have Material demand multiple-microorganism co-incubation could generate, thus microbial co culture become industry, agricultural, medicine, food and The hot issue in the fields such as environmental protection, and have become the important method for improving yield or finding novel substance.

The complicated utilization of existing microorganism is mostly the direct mixing of several strains, and this mode obtains bacterial strain can not be steady It is fixed to exist, easily lead to that flora is unbalance and strain unification, the product quality of this composite bacteria agent remain to be discussed.

In addition, current symbiosis composite bacteria fermentation technique is complicated for operation, higher cost, while strain compound process is more Cumbersome, requirement due to each bacterial strain to nutrition with environment is different, it is difficult to Human disturbance control, especially for oxygen demand and The different bacterial strain of tolerance degree, the symbiosis culture of the prior art are confined to same type different genera microorganism syntrophism, mesh Before, require different aerobic bacterias, anaerobic bacteria and the artificial induction of facultative anaerobic bacteria and symbiosis culture technique not to obtain oxygen environment To breakthrough.Meanwhile existing composite bacteria stability is poor, it is desirable that metastable Reproduction Conditions；Environmental suitability is poor, can only be used to Strain induction screening environmental condition, practical ranges are small, and it is unbalance that the minor alteration of environment is likely to result in flora, strain list One changes.In practical application, the poor processing effect of independent aerobic bacteria or independent anaerobic bacteria, the prior art is mostly aerobic bacteria, anaerobic bacteria It is successively applicable in, is repeatedly handled, the process flow is long, and project cost is big.Existing complex micro organism fungicide practical application effect Not significant, the strain dosage needed is excessive.Therefore, a kind of bacterial strain symbiosis different with tolerance degree for oxygen content requirements is developed The technical method of culture has great importance.

In view of this, the present invention is specifically proposed.

Summary of the invention

The first object of the present invention is to provide a kind of abductive approach of microbe symbiotic culture, to alleviate in the prior art Lacking one kind can be by the method for the bacterial strain symbiosis culture different with tolerance degree to oxygen content requirements.

The second object of the present invention is to provide a kind of microbe symbiotic culture microbial inoculum, lacks one in the prior art to alleviate The composite bacteria agent of the kind bacterial strain symbiosis different with tolerance degree to oxygen content requirements.

The third object of the present invention is to provide the application of mentioned microorganism symbiosis culture microbial inoculum.

In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:

A kind of abductive approach of microbe symbiotic culture, comprising the following steps:

First kind bacterium and the second class bacterium by induction processing are mixed, super oxygen is added with the first preset interval time Compound mutase, and, to preset O when incrementss improve culture₂20%-22% is measured, microbe symbiotic culture microbial inoculum is obtained；

The induction processing includes: that superoxides is added with the first preset interval time in the second class bacterium incubation Mutase, and, O when predetermined amount improves culture is increased with the second preset interval time₂Measure 5%-20%；

The first kind bacterium is aerobic bacteria or facultative anaerobic bacteria；

The second class bacterium is at least one of facultative anaerobic bacteria or anaerobic bacteria；

Wherein, the first kind bacterium and the second class bacterium are different.

Further, the culture medium of aerobic bacteria, facultative anaerobic bacteria or mixed culture independently includes: 5-10g/L red Sugar, 5-15g/L peptone, 1-5g/L powdered beef and 3-7g/L sodium chloride, pH7.2-7.4；

Preferably, the culture medium of the anaerobic bacteria includes: 15-25g/L pancreas casein peptone, 3-7g/L NaCl, 1-3g/L Sodium thioglycolate, 0.5-2g/L sodium formaldehyde sulphoxylate, 0.001-0.005g/L methylene blue and 5-10g/L brown sugar, pH7.4- 7.6。

Further, first preset interval time is 1-5h；

Preferably, the additional amount of the superoxide dismutase is 0.005%-0.5% (w/v, 20000IU/g), preferably For 0.005%-0.1% (w/v, 20000IU/g), further preferably 0.005%-0.05% (w/v, 20000IU/g)；

Preferably, the default incrementss are 1%-8%/day, preferably 1%-5%；

Preferably, second preset interval time is 2-3 days；

Preferably, the predetermined amount is 1%-5%；

It preferably, further include by CO in the abductive approach₂Content is reduced to the step of 0.02%-0.5% from 9%-11% Suddenly.

Preferably, when aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria independently include two or more bacterial strains, by the bacterial strain It co-cultures 2-3 days, obtains aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria.

Preferably, the bacteria concentration that co-culture system is added in first kind bacterium or the second class bacterium independently is 10⁶-10⁸cfu/ Ml, inoculum concentration 2-5%.

Further, the aerobic bacteria includes acetobacter xylinum, bacillus subtilis, nitrobacteria, acetobacter or fixed nitrogen At least one of bacterium.

Further, the anaerobic bacteria includes that clostridium butyricum, Bacillus acidi lactici, Bifidobacterium, methanogen or denitrification are thin At least one of bacterium.

Further, the facultative anaerobic bacteria includes Leuconostoc mesenteroides, saccharomycete, bacillus amyloliquefaciens, Bei Laisi Bacillus, Bacillus cercus, bacillus pumilus, lactobacillus plantarum, candida utili, bacillus licheniformis or yellowish-brown At least one of pseudomonad.

The microbe symbiotic culture microbial inoculum that above-mentioned abductive approach obtains.

Above-mentioned abductive approach or microbe symbiotic culture microbial inoculum are in following A)-D) application in any one:

A) effective component of animal or plant is extracted；

B microbial-bacterial fertilizer) is prepared；

C) degradation noxious material；

D) degradation larger molecular organics matter.

Compared with prior art, the invention has the benefit that

The abductive approach of microbe symbiotic culture provided by the invention overcomes different with tolerance degree to oxygen content requirements Bacterial strain, even obligate aerobic bacteria and obligate anaerobe, are unable to the technical issues of symbiosis culture obtains composite bacteria agent.According to target Demand of the bacterial strain to oxygen content is classified, and the close bacterial strain of oxygen demand is first merged culture and obtains several bacterial strain groups, from need The minimum bacterial strain group of oxygen amount starts the induction of symbiosis culture, by the amount of bioactivity superoxide dismutase in guarantee system and The oxygen content in condition of culture is gradually increased to promote the adaptability of system, when oxygen content reaches a certain concentration, oxygen is added Bacterial strain group similar in content demand, then pass through the amount of superoxide dismutase in guarantee system and be gradually increased in condition of culture Oxygen content promotes system to the adaptability of more oxygen content, repeats above-mentioned operation, until all bacterial strain groups, i.e. target Bacterial strain whole symbiosis culture is completed, and can obtain microbe symbiotic culture bacterium under field conditions (factors) with the coupled growth of normal table Agent.This method is simple and easy to operate, and production cost is low, and universality is wide, can satisfy a variety of different symbiosis culture demands.This method In each bacterial strain pass through during the cultivation process constantly adjustment can achieve stable balance commensalism, the stability of composite flora Good, adaptable, the phenomenon that being not in unbalance flora and unification, obtained microbe symbiotic culture microbial inoculum can be used as production Product or conservation directly use.

Microbe symbiotic culture microbial inoculum provided by the invention, which has, to be stablized, and strong advantage is adapted to, and is not in that flora loses It is the phenomenon that weighing apparatus and unification, at low cost, can be by a large amount of production of fermenting, preparation cost is low, has a wide range of application, and is suitble to each row Industry actual production uses.

Detailed description of the invention

Fig. 1 is the testing result of bacillus subtilis 16S rRNA in embodiment 1；

Fig. 2 is the testing result of methanogen 16S rRNA in embodiment 1；

Fig. 3 is the qualification result of bacillus pumilus in embodiment 2；

Fig. 4 is that denitrifying bacteria is based on 16S rDNA testing result progress phylogenetic tree analysis result in embodiment 2；

Fig. 5 is the qualification result of acetobacter in embodiment 3；

Fig. 6 is the qualification result of lactobacillus plantarum in embodiment 4；

Fig. 7 is the qualification result of bacillus subtilis in embodiment 4.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.

In the present invention, if without particularly illustrating, all embodiments mentioned in this article and preferred implementation method It can be combined with each other to form new technical solution.

In the present invention, if without particularly illustrating, all technical characteristics and preferred feature mentioned in this article can be with Intercombination forms new technical solution.

In the present invention, if related each component or its preferred ingredient can be combined with each other shape without particularly illustrating The technical solution of Cheng Xin.

In the present invention, unless otherwise indicated, numberical range " a~b " indicates the contracting of any real combinings between a to b Sketch form shows that wherein a and b is real number.Such as numberical range " 6~22 " indicate herein all listed " 6~22 " it Between whole real numbers, " 6~22 " be these combinations of values breviary indicate.

" range " disclosed in this invention can be respectively one or more lower limits and one in the form of lower and upper limit A or multiple upper limits.

In the present invention, unless otherwise indicated, each reaction or operating procedure can be carried out sequentially, can not also be in sequence It carries out.Preferably, reaction method herein is that sequence carries out.

Unless otherwise indicated, profession used herein and meaning phase known to scientific term and one skilled in the art Together.In addition, any method similar to or equal to what is recorded or material can also be applied in the present invention.

Wherein, induction processing includes: that superoxides is added with the first preset interval time in the second class bacterium incubation Mutase, and, O when predetermined amount improves culture is increased with the second preset interval time₂Measure 5%-20%；

Wherein, first kind bacterium is aerobic bacteria or facultative anaerobic bacteria；Second class bacterium be in facultative anaerobic bacteria or anaerobic bacteria extremely Few one kind；The first kind bacterium and the second class bacterium are different.

When aimed strain is aerobic bacteria and anaerobic bacteria, first aerobic bacteria and anaerobic bacteria are separately cultivated, anaerobic bacteria exists 10%H₂, 10%CO₂, 80%N₂Under the conditions of cultivated, when if a variety of anaerobic bacterias, a variety of anaerobic bacterias are first subjected to mixing training The stabilization for ensuring system for 2-3 days is supported, 0.005-0.05%SOD (20000IU/g) is added every 2h, cultivates 2-3 days, ventilation changes For 2%O₂, 8%CO₂, 80% N₂It is further cultured for 2-3 days, amount of oxygen increases to 3-5%, is spaced 2-3 days, amount of oxygen increases to 7- 10%, increase by 2% daily later, when to 15% or so, cultivate 2-3 days, cultured aerobic bacteria is added, later every 4h addition Same amount of SOD is cultivated 2-3 days, stops that SOD is added, and increases by 2% every 12h oxygen-supply quantity, until 20-22%, continuously cultivates 2- 3 days, period CO₂Content constantly decline, eventually arrive at the content in air, obtain stable in the natural environment aerobic bacteria and Anaerobic bacteria symbiosis culture microbial inoculum.

When aimed strain is aerobic bacteria and facultative anaerobic bacteria, first aerobic bacteria and facultative anaerobic bacteria are separately cultivated, Facultative anaerobic bacteria is in 3-5%O₂, 10%CO₂, 85%N₂Under the conditions of cultivated, first will be a variety of when if a variety of facultative anaerobic bacterias Facultative anaerobic bacteria, which carries out mixed culture, ensures the stabilization of system for 2-3 days, and 0.005-0.05%SOD (20000IU/ is added every 4h G), it cultivates 2-3 days, amount of oxygen increases to 7-10%, increases by 2% daily later, when to 15% or so, cultivates 2-3 days, addition is trained Same amount of SOD is added every 4h later in the aerobic bacteria supported, and cultivates 2-3 days, stops that SOD is added, increases every 12h oxygen-supply quantity Add 2%, until 20-22%, continuous culture 2-3 days, period CO₂Content constantly decline, eventually arrive at the content in air, obtain To aerobic bacteria stable in the natural environment and facultative anaerobic bacteria symbiosis culture microbial inoculum.

When aimed strain is aerobic bacteria, facultative anaerobic bacteria and anaerobic bacteria, first by aerobic bacteria, facultative anaerobic bacteria and anaerobic bacteria It is separately cultivated, anaerobic bacteria is in 10%H₂, 10%CO₂, 80%N₂Under the conditions of cultivated, when if a variety of anaerobic bacterias, first will A variety of anaerobic bacterias, which carry out mixed culture, ensures the stabilization of system for 2-3 days, and 0.005-0.05%SOD (20000IU/ is added every 2h G), it cultivates 2-3 days, ventilation is changed to 2%O₂, 8%CO₂, 80%N₂It is further cultured for 2-3 days, amount of oxygen increases to 3-5%, is spaced 2-3 It, is added facultative anaerobic bacteria, and amount of oxygen increases to 7-10%, increases by 2% daily later, when to 15% or so, cultivates 2-3 days, Cultured aerobic bacteria is added, same amount of SOD is added every 4h later, cultivates 2-3 days, stops that SOD is added, it is logical every 12h Oxygen amount increases by 2%, until 20-22%, continuous culture 2-3 days, period CO₂Content constantly decline, eventually arrive in air Content obtains aerobic bacteria stable in the natural environment, facultative anaerobic bacteria and anaerobic bacteria symbiosis culture microbial inoculum.

When aimed strain is facultative anaerobic bacteria and anaerobic bacteria, first facultative anaerobic bacteria and anaerobic bacteria are separately cultivated, Anaerobic bacteria is in 10%H₂, 10%CO₂, 80%N₂Under the conditions of cultivated, when if a variety of anaerobic bacterias, first by a variety of anaerobic bacterias into Row mixed culture ensures the stabilization of system for 2-3 days, and 0.005-0.05%SOD (20000IU/g) is added every 2h, cultivates 2-3 It, ventilation is changed to 2%O₂, 8%CO₂, 80%N₂It is further cultured for 2-3 days, amount of oxygen increases to 3-5%, is spaced 2-3 days, is added simultaneous Property anaerobic bacteria, amount of oxygen increases to 7-10%, increases by 2% daily later, when to 15% or so, cultivates 2-3 days, later every 4h Same amount of SOD is added, cultivates 2-3 days, stops that SOD is added, increases by 2% every 12h oxygen-supply quantity, until 20-22%, continuous to train It supports 2-3 days, period CO₂Content constantly decline, eventually arrive at the content in air, obtain stable facultative in the natural environment Anaerobic bacteria and anaerobic bacteria symbiosis culture microbial inoculum.

Superoxide dismutase is provided to symbiosis cultivating system and is gradually increased from the above, it can be seen that the present invention is utilized Oxygen content in culture environment improves the adaptability of strain, and in symbiosis cultivating system different strains reaches a balance and stability State so that different types of strain realizes symbiosis culture in natural conditions.

In being preferably carried out mode, the culture medium of aerobic bacteria, facultative anaerobic bacteria or mixed culture independently includes: 5- 10g/L brown sugar, 5-15g/L peptone, 1-5g/L powdered beef and 3-7g/L sodium chloride, pH7.2-7.4.It is understood that training Supporting base is fluid nutrient medium, and solvent is sterile water.It should be noted that brown sugar it is typical but non-limiting for 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L；Peptone it is typical but non-limiting for 5g/L, 7g/L, 9g/L, 11g/L, 13g/L or 15g/L；Typical but non-limiting powdered beef is 1g/L, 2g/L, 3g/L, 4g/L or 5g/L；Sodium chloride is typical but non-limiting It is 3g/L, 4g/L, 5g/L, 6g/L or 7g/L.

In being preferably carried out mode, the culture medium of anaerobic bacteria includes: 15-25g/L pancreas casein peptone, 3-7g/L NaCl, 1-3g/L sodium thioglycolate, 0.5-2g/L sodium formaldehyde sulphoxylate, 0.001-0.005g/L methylene blue and 5-10g/L Brown sugar, pH7.4-7.6.It is understood that culture medium is fluid nutrient medium, solvent is sterile water, and methylene blue is indicator, anaerobic Shi Hongse, it is blue when aerobic.It should be noted that pancreas casein peptone it is typical but it is unrestricted for 15g/L, 17g/L, 20g/L, 22g/L or 25g/L；NaCl typical but non-limiting is 3g/L, 4g/L, 5g/L, 6g/L or 7g/L；Sodium thioglycolate allusion quotation Type but it is unrestricted be 1g/L, 2g/L or 3g/L；Typical but non-limiting sodium formaldehyde sulphoxylate is 0.5g/L, 1g/ L, 1.5g/L or 2g/L；Methylene blue it is typical but non-limiting for 0.001g/L, 0.002g/L, 0.003g/L, 0.004g/L, 0.005g/L；Typical but non-limiting brown sugar is 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L.

In above two culture medium, brown sugar is without the highly refined sucrose of process, except providing carbon source, nitrogen source for microorganism Outside, there are also various trace elements and minerals in the red pool, separately have riboflavin, amino acid, vitamin etc., are providing nutrition for biology While substance, has and adjust osmotic pressure inside and outside organism, boost metabolism, quickly activate suspend mode bacterium, improve microbial activity The effects of ability.

In being preferably carried out mode, the first preset interval time is 1-5h.In order to guarantee to contain always in syntaxial system Superoxide dismutase improves the adaptability of bacterial strain, supplements superoxide dismutase every 1-5h, the time is preferably 2-4h.

In being preferably carried out mode, the additional amount of superoxide dismutase be 0.005%-0.5% (w/v, 20000IU/g), preferably 0.005%-0.1% (w/v, 20000IU/g), further preferably 0.005%-0.05% (w/ V, 20000IU/g).

Superoxide dismutase (Superoxide Dismutase, SOD) is antioxidase important in organism, extensively It is distributed in various organisms, such as animal, plant, microorganism etc..SOD has special physiological activity, is removed in organism The primary substance of free radical.SOD is naturally occurring superoxide radical clearing factor in body, can be harmful by reaction Superoxide radical is converted into hydrogen peroxide.Although hydrogen peroxide is still to the harmful active oxygen of body, intracorporal hydrogen peroxide Enzyme (CAT) and peroxidase (POD) can be broken down into completely harmless water immediately.In this way, three kinds of enzymes just constitute one Complete biological chain.Anaerobic bacteria lacks SOD and cytochrome oxidase into the cell, most of to also lack catalase, passes through Guarantee that the presence of superoxide dismutase in cultivating system reduces oxygen to the oxytolerant of anaerobic bacteria toxic action and/or facultative anaerobic bacteria Ability.

SOD theory thinks that strictly anaerobic microorganism is not to be killed by gaseous oxygen, but certain due to that cannot release The toxicity of oxygen metabolism product and it is dead.

During hydrogen reduction is water, certain toxic intermediate products can be formed, for example, hydrogen peroxide (H₂O₂), super oxygen Anion (O₂) etc..Superoxide anion is active oxygen, has the property of molecule and ion concurrently, and reagency is extremely strong, extremely unstable, can Film and important biomolecule macromolecular are destroyed, microorganism is caused to poison or lethal.Facultative anaerobic bacteria and aerobic bacteria, which have, degrades these The enzyme of product, such as catalase, peroxidase, superoxide dismutase (SOD), but metabolic degradation ability not phase Together, strict anaerobes shortage SOD, therefore the ultra-oxygen anion free radical murder by poisoning easily easily generated in organism is lethal.

By providing suitable SOD oxygen-resistant ability for improving strain, side during symbiosis cultivating system is induced and established It helps between strain and reaches a kind of symbiosis balance.

In being preferably carried out mode, presetting incrementss is 1%-8%/day, preferably 1%-5%.

In being preferably carried out mode, the second preset interval time is 2-3 days.

In being preferably carried out mode, predetermined amount 1%-5%.

It further include by CO in being preferably carried out mode, in abductive approach₂Content is reduced to 0.02%- from 9%-11% 0.5% the step of.

In being preferably carried out mode, aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria independently include two or more bacterial strains When, bacterial strain is co-cultured 2-3 days, aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria are obtained.It is excellent when the strain quantity of target is more Bacterial strain is divided into aerobic bacteria, facultative anaerobic bacteria and anaerobic bacteria three classes and is mixed respectively by choosing, in the structure of symbiosis cultivating system In building, bacterium solution, i.e. anaerobic bacteria, facultative anaerobic bacteria and aerobic bacteria three times is only at most added, reduces system in this way and strain is added Number reduces the risk of microbiological contamination.

In being preferably carried out mode, the initial number ratio of first kind bacterium and the second class bacterium is 1:0.5-1.5.It needs to illustrate , the specific strain, first kind bacterium or the second class bacterium that are related in the present invention, when carrying out culture operation, mixed bacterium amount It is essentially identical, it is preferably 10 according to concentration⁶-10⁸Cfu/ml, inoculum concentration are that the amount of 2-5% is added.Such as can with but not It is limited to, the initial incremental amount of first kind bacterium and the second class bacterium when symbiosis cultivating system is added is 10 according to concentration⁶-10⁸cfu/ Ml, inoculum concentration are that the amount of 2-5% is added；When first kind bacterium and/or the second class bacterium are made of two or more strains, two kinds It is 10 that the co-cultivation of the above strain, which is also according to concentration,⁶-10⁸Cfu/ml, inoculum concentration are that the amount of 2-5% is added, culture balance The first kind bacterium and/or the second class bacterium obtained afterwards, first kind bacterium and the second class bacterium are first when symbiosis cultivating system is added later Beginning additional amount also according to concentration be 10⁶-10⁸Cfu/ml, inoculum concentration are that the amount of 2-5% is added.

In being preferably carried out mode, aerobic bacteria include but is not limited to acetobacter xylinum, bacillus subtilis, nitrobacteria, At least one of acetobacter or nitrogen-fixing bacteria.

In being preferably carried out mode, anaerobic bacteria includes but is not limited to clostridium butyricum, Bacillus acidi lactici, Bifidobacterium, produces first At least one of alkane bacterium or denitrifying bacteria.

In being preferably carried out mode, facultative anaerobic bacteria includes but is not limited to Leuconostoc mesenteroides, saccharomycete, solution starch bud Spore bacillus, Bei Laisi bacillus, Bacillus cercus, bacillus pumilus, lactobacillus plantarum, candida utili, lichens At least one of bacillus or yellowish-brown pseudomonad.

The present invention provides the microbe symbiotic culture microbial inoculum that above-mentioned abductive approach obtains.

A) effective component of animal or plant is extracted；

B microbial-bacterial fertilizer) is prepared；

C) degradation noxious material；

D) degradation larger molecular organics matter.

Symbiosis culture abductive approach provided by the invention can will realize the oxygen demand strain different with tolerance degree Symbiosis culture, the abductive approach and obtained microbial inoculum can be applied in the various scenes of production and living:

The effective component of animal or plant is extracted, for example, the extraction and preparation of natural products, in plant and animal The abundant extraction of natural drug in medicinal material, microbe symbiotic culture microbial inoculum can preferably destroy cell, so that effective component It is released；The full ingredient high value of the production of agricultural and sideline product, various agricultural product utilizes；Prepare high value feed etc..

Microbial-bacterial fertilizer is prepared, various strains can be with one balance commensalism of syntrophism and holding, the bacterial manure of preparation Also more efficient.

Degradation noxious material, is handled for water, the soil etc. of the pollutions such as pesticide, heavy metal, petroleum, at low cost, behaviour Make simple.

Since the symbiosis culture of different oxygen demand strains under field conditions (factors) may be implemented in the abductive approach, so, it can be with The tedious steps for overcoming the composite bacteria that oxygen demand is close in the prior art to need repeatedly to compound, and, oxygen demand difference is biggish The problem of the impossible co-incubation of composite bacteria.Different strain collective effect, realization synchronize to target macromolecule organic substance into Row degradation has more significant effect for the degradation of target macromolecule organic substance.

Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only It is used, is but should not be understood as present invention is limited in any form for being described in more detail.

Aerobic bacteria and the culture medium of facultative anaerobic bacteria independently are 7g/L brown sugar, 10g/L albumen in following embodiments Peptone, 3g/L powdered beef and 5g/L sodium chloride, pH7.3, are denoted as the first culture medium.

The culture medium of anaerobic bacteria is 20g/L pancreas casein peptone, 5g/L NaCl, 2g/L sodium thioglycolate, 1g/L formaldehyde Sodium bisulphite, 0.002g/L methylene blue and 7g/L brown sugar, pH7.5 are closed, the second culture medium is denoted as.

Embodiment 1

Aerobic bacteria: bacillus subtilis；

Anaerobic bacteria: methanogen；

1, collect strain, enrichment culture in culture medium: the first culture medium of aerobic bacteria, 37 DEG C, biochemical cultivation case is routinely trained It supports；Anaerobic bacteria is passed through mixed gas: 10% H in the second culture medium anaerobism work station₂, 10%CO₂, 80%N₂.Cultivate 2-3 It.

2, take 1 bacterium solution that sterile water is added successively to be diluted to 10⁴Cfu/ml, 10⁶Cfu/ml, 10⁸cfu/ml.Each dilution Degree takes 0.1-0.2mL to apply plate, and 3 parallel, cultivates 2-3 days under the conditions of 37 DEG C, different according to colonial morphology feature picking Single colonie, scribing line purifying.

3, the purified bacterium colony of picking is inverted culture 3-4d, scrapes thallus, does molecular biology and Morphological Identification.

4,0.005-0.05%SOD (20000IU/g) is added into anaerobism bacterium solution, after being cultivated 2-3 days in anaerobism work station, 1-2%O is passed through into anaerobism work station₂, 8%CO₂, 80%N₂, continuing after cultivating 2-3d, oxygen-supply quantity is slowly increased to 3-5%, Continue to cultivate 3d.The identical bioactivity SOD of 0.005-0.05% is added every 2h during this.

5, oxygen-supply quantity increases to 7-10% after 2-3 days, cultivates 2-3 days, hereafter increases by 2% every oxygen-supply quantity for 24 hours, residual air Body component is constant, until oxygen-supply quantity increases to 15%.0.005-0.05%SOD is added every 4h during this.

6,5 bacterium solutions are taken, cell concentration 10 is configured to⁶-10⁸Cfu/ml, by inoculum concentration 2-5% in 150-250ml In one culture medium, is cultivated under the conditions of amount of oxygen 15% 2-3 days, 0.005-0.05%SOD during which is added every 4h.According to 2-5% Inoculum concentration be added concentration 10⁶-10⁸The aerobic bacterium solution of cfu/ml after culture 2-3 days, stops that SOD is added, increase every 12h logical Oxygen amount 2%, until leading to oxygen to 20-21%, CO₂Content is reduced to 0.03-0.5%.

7, above 6 bacterium solutions continuously cultivate 2-3 days under the conditions of biochemical cultivation case in the first culture medium, obtain symbiosis Cultivating system.

8, it takes 7 bacterium solutions to do molecular biology and Morphological Identification, as a result compares with 3 results, determine strain.

The qualification result of bacterial strain is as depicted in figs. 1 and 2, testing result of the Fig. 1 for bacillus subtilis 16S rRNA, M: maker；1: the qualification result of bacillus subtilis before Fiber differentiation；2: the qualification result of bacillus subtilis after Fiber differentiation. Fig. 2 is the testing result of methanogen 16S rRNA, 1 and 4:maker；2: the qualification result of methanogen before Fiber differentiation；3: The qualification result of methanogen after Fiber differentiation.

Illustrate that abductive approach provided by the invention successfully realizes the symbiosis culture of bacillus subtilis and maternity leave alkane bacterium, obtains The microbial inoculum of symbiosis culture under two kinds of bacterial strain natural conditions.

The primer sequence used in detection is as shown in table 1 below:

Table 1

	Sequence (5 ' -3 ')	Sequence number
			Bacillus subtilis-F	AGAGTTTGATCCTGGCTCAG	SEQ ID NO.1
Bacillus subtilis-R	AAGGAGGTGATCCAGCCGCA	SEQ ID NO.2
			Methanogen-F	TCCGGTTGATCCCGCCGG	SEQ ID NO.3
Methanogen-R	CCGTCAATTCCTTTGAGTTT	SEQ ID NO.4

Embodiment 2

Facultative anaerobic bacteria: bacillus amyloliquefaciens, Bei Laisi bacillus, Bacillus cercus and bacillus pumilus；

Anaerobic bacteria: denitrifying bacteria；

1, strain is collected, enrichment culture in culture medium: the first culture medium of facultative anaerobic bacteria is placed in three gas incubators In, it is passed through mixed gas: 5%O₂, 10%CO₂, 80%N₂；Anaerobic bacteria is passed through gaseous mixture in the second culture medium anaerobism work station Body: 10%H₂, 10%CO₂, 80%N₂.Culture 2-3 days.

4, concentration 10 is measured by the inoculation of 2-5%⁶-10⁸Each bacterium solution of cfu/ml, merge all amphimicrobian culture bacterium solutions in The first culture medium of 150-250ml is cultivated 2-3 days under optimum conditions.

5, to anaerobism bacterium solution (according to concentration 10⁶-10⁸Cfu/ml and inoculum concentration 2-5% cultivate to obtain) in be added 0.005- 0.05%SOD (20000IU/g) is passed through 1-2%O into anaerobism work station after cultivating 2-3 days in anaerobism work station₂, 8%CO₂, 80%N₂, continue after cultivating 2-3d, oxygen-supply quantity is slowly increased to 3-5%, continues to cultivate 3d.It is added during this every 2h The identical bioactivity SOD of 0.005-0.05%.

6, to the first culture medium is added in 5,0.005-0.05%SOD is added, incubator is passed through mixed gas 3-5%O₂, 10%CO₂, 85%N₂.After 2-3 days, the 4 amphimicrobian bacterium solution (concentration of facultative anaerobic bacteria are added according to the inoculum concentration of 2-5% 10⁶-10⁸Cfu/ml), oxygen-supply quantity increases to 7-10%, cultivates 2-3 days, hereafter increases by 2% every oxygen-supply quantity for 24 hours, remaining gas Component is constant, until oxygen-supply quantity increases to 15%.0.005-0.05%SOD is added every 4h during this.

7, after cultivating 2-3 days, stop that SOD is added, increase oxygen-supply quantity 2% every 12h, up to logical oxygen to 20-21%, CO₂ Content is reduced to 0.03-0.5%.

8, above 7 bacterium solutions continuously cultivate 2-3 days under the conditions of biochemical cultivation case in the first culture medium, obtain symbiosis Cultivating system.

9, it takes 8 bacterium solutions to do molecular biology and Morphological Identification, as a result compares with 3 results, determine strain.

The qualification result of bacillus pumilus is as shown in Figure 3, wherein M:maker；1: the mirror of bacillus pumilus before inducing Determine result；3: the qualification result of bacillus pumilus after induction.

16S rDNA detection, testing result such as SEQ ID NO.9 institute are carried out to denitrifying bacteria (Strain Designation LZ14) Show, phylogenetic tree analysis is carried out based on 16S rDNA testing result, as a result as shown in figure 4, identification obtains denitrifying bacteria.

Successful identification obtains to be identified respectively to bacillus amyloliquefaciens, Bei Laisi bacillus, Bacillus cercus Bacterial strain before induction and after induction.The result shows that above-mentioned bacterial strains are successfully realized symbiosis culture by abductive approach provided by the invention, Obtain the mix bacterium agent grown under natural conditions.

Detection primer is as shown in table 2 below:

Table 2

	Sequence (5 ' -3 ')	Sequence number
			Bacillus pumilus-F	AGAGTTTGATCCTGGCTTTAG	SEQ ID NO.5
Bacillus pumilus-R	ACGGCTACCTTGTTACGACTT	SEQ ID NO.6
			Denitrifying bacteria-F	AGAGTTTGATCCTGGCTCAG	SEQ ID NO.7
Denitrifying bacteria-R	GGTTACCTTGTTACGACTT	SEQ ID NO.8

TGCAGTCGAGCGGATGAAGGGAGCTTGCTCTCTGATTCAGCGGC GGACGGGTGAGTAATGCCTAGGA ATCTGCCTGATAGTGGGGGACAAC GCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGG GAC CTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAG TTGGTGAGGTAACGGCTCACCAAGGCGACG ATCCGTAACTGGTCTGA GAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTAC GGGAGGCAGC AGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCC AGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTA AAGCACAAG TTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCG ACGAATAAGCACCGGCT AACTTCGTGCCAGCAGCCGCGGTAATACGA AGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTA GG TGGGTTAAGTTGAATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCA TCCAAAACTGGCAAGCTAGAGTATG GCAGAGGGTGGTGGAACCTGTG TAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGC GACCA CCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGC AAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGTCGACT AGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGT CGACCGCCTGG GGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTG ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGAATTCGA AGCAACG CGAAGAACCTTACCAGGCCTTGACATGCAGAGAACCCAGAGATGGAT TGGTGCCTTCGGGAACTCTG ACACAGGTGCTGCATGGCTGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTT GTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCC GGTGACAAACCGGAGGAAGGTGGGGATG ACGTCAAGTCATCATGGCC CTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTT GCCAAGC CGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCG GATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGA ATCGCTAGTAATC GTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCA TGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAA C(SEQ ID NO.9)

Embodiment 3

Aerobic bacteria: acetobacter；

Facultative anaerobic bacteria: bacillus licheniformis；

Anaerobic bacteria: denitrifying bacteria；

1, collect strain, enrichment culture in culture medium: the first culture medium of aerobic bacteria, 37 DEG C, biochemical cultivation case is routinely trained It supports；The first culture medium of facultative anaerobic bacteria, is placed in three gas incubators, is passed through mixed gas: 5%O₂, 10%CO₂, 80% N₂；Anaerobic bacteria is passed through mixed gas in the second culture medium anaerobism work station: 10%H₂, 10%CO₂, 80%N₂.Culture 2-3 days.

2, take 1 bacterium solution that sterile water is added successively to be diluted to 10⁴Cfu/ml, 10⁶Cfu/ml, 10⁸cfu/ml.Each dilution Degree takes 0.1-0.2m L to apply plate, and 3 parallel, cultivates 2-3 days under the conditions of 37 DEG C, not according to colonial morphology feature picking Same single colonie, scribing line purifying.

4, to anaerobism bacterium solution (according to concentration 10⁶-10⁸Cfu/ml and inoculum concentration 2-5% cultivate to obtain) in be added 0.005- 0.05%SOD (20000IU/g) is passed through 1-2%O into anaerobism work station after cultivating 2-3 days in anaerobism work station₂, 8%CO₂, 80%N₂, continue after cultivating 2-3d, oxygen-supply quantity is slowly increased to 3-5%, continues to cultivate 3d.It is added during this every 2h The identical bioactivity SOD of 0.005-0.05%.

5, to the first culture medium is added in 4,0.005-0.05%SOD is added, incubator is passed through mixed gas 3-5%O₂, 10%CO₂, 85%N₂.After 2-3 days, it is 10 that concentration, which is added, according to the inoculum concentration of 2-5%⁶-10⁸The amphimicrobian bacterium solution of cfu/ml, Oxygen-supply quantity increases to 7-10%, cultivates 2-3 days, hereafter increases by 2% every oxygen-supply quantity for 24 hours, and remaining gas component is constant, until logical Oxygen amount increases to 15%.0.005-0.05%SOD is added every 4h during this.

6,5 bacterium solutions are taken, cell concentration 10 is configured to⁶-10⁸Cfu/ml, by inoculum concentration 2-5% in 150-250ml In one culture medium, is cultivated under the conditions of amount of oxygen 15% 2-3 days, 0.005-0.05%SOD during which is added every 4h.According to inoculum concentration It is 10 that concentration, which is added, in 2-5%⁶-10⁸The aerobic bacterium solution 2-5% inoculum concentration of cfu/ml after culture 2-3 days, stops that SOD is added, every 12h increases oxygen-supply quantity 2%, until leading to oxygen to 20-21%, CO₂Content is reduced to 0.03-0.5%.

The qualification result of acetobacter is as shown in Figure 5, wherein M:maker；2 and 3: the identification knot of acetobacter before inducing Fruit；4 and 5: the qualification result of acetobacter after induction.

16S rDNA detection is carried out to bacillus licheniformis, testing result is as shown in SEQ ID NO.14, in the website NCBI Blast comparison is carried out, strain Bacillus licheniformis (accession number: JX025165) homology 100% is added with original.

Identical in the identification mode such as embodiment 2 of denitrifying bacteria, last successful identification obtains denitrifying bacteria.

The result shows that abductive approach provided by the invention successfully obtains the mixing of symbiosis culture under three kinds of bacterial strain natural conditions Microbial inoculum.

Detection primer is as shown in table 3 below:

Table 3

	Sequence (5 ' -3 ')	Sequence number
			Acetobacter-F	AGAGTTTGATCCTGGCAG	SEQ ID NO.10
Acetobacter-R	GGGTACCTTGTTACGACTT	SEQ ID NO.11
			Bacillus licheniformis-F	AGAGTTTGATCCTGGCTCAG	SEQ ID NO.12
Bacillus licheniformis-R	CTACGGCTACCTTGTTACGA	SEQ ID NO.13

TGTCACTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTC GGGTGTTACAAACTCTCGTGGTG TGACGGGCGGTGTGTACAAGGCCC GGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCC AG CTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGA TTTGTGGGATTGGCTTAGCCTCGCGGCTTC GCTGCCCTTTGTTCTGCCC ATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGAC GTCATCC CCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTG CCCAACTGAATGCTGGCAACTAAGATCAAGGGTT GCGCTCGTTGCGG GACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACC ACCTGTCACTCTGC CCCCGAAGGGGAAGCCCTATCTCTAGGGTTGTCA GAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGA ATTAAAC CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTC AGTCTTGCGACCGTACTCC CCAGGCGGAGTGCTTAATGCGTTTGCTGC AGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTA CG GCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGC GCCTCAGCGTCAGTTACAGACCAG AGAGTCGCCTTCGCCACTGGTGT TCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTC CT CTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGA GCCGGGGGCTTTCACATCAGACTTAAGAA ACCGCCTGCGCGCGCTTT ACGCCCAATAATTCCCGGAAACGCTTGCCACCTACGTATTACCGCGGC TGCTGGCA CGTAGTTTAGCCGTGGCTTTCTGGGTTAGGTACCGTCAAG GTGCCGCCCTATTCGAACGGTACTGTCTCCTACAC AGAGTTTTACGAT CCGAAAACCTTCATCACTCACGCGGGCGTTGCTCCGTCAGAACTTTCG T(SEQ ID NO.14)

Embodiment 4

Aerobic bacteria: acetobacter, bacillus subtilis；

Facultative anaerobic bacteria: bacillus amyloliquefaciens, Bei Laisi bacillus, Bacillus cercus, bacillus pumilus, plant Object lactobacillus, candida utili, bacillus licheniformis and yellowish-brown pseudomonad；

Anaerobic bacteria: methanogen, denitrifying bacteria；

4,2-5% inoculum concentration, concentration 10 are pressed⁶-10⁸Cfu/ml merges all aerobic culture bacterium solutions in 150-250ml first Culture medium merges all amphimicrobian culture bacterium solutions in the first culture medium of 150-250ml, merges all anaerobic bacteria bacterium in 150- In the second culture medium of 250ml, cultivated 2-3 days under respective suitable condition.

7,6 bacterium solutions are taken, cell concentration 10 is configured to⁶-10⁸Cfu/ml, by inoculum concentration 2-5% in 150-250ml In one culture medium, is cultivated under the conditions of amount of oxygen 15% 2-3 days, 0.005-0.05%SOD during which is added every 4h.According to inoculum concentration Concentration 10 is added in 2-5%⁶-10⁸Cfu/ml aerobic bacterium solution inoculum concentration after culture 2-3 days, stops that SOD is added, increase every 12h logical Oxygen amount 2%, until leading to oxygen to 20-21%, CO₂Content is reduced to 0.03-0.5%.

The qualification result of lactobacillus plantarum is as shown in Figure 6, wherein M:maker；3: the identification knot of lactobacillus plantarum before inducing Fruit；1: the qualification result of lactobacillus plantarum after induction.

Bacillus subtilis is identified, primer is in the same manner as in Example 1, as a result as shown in Figure 7, wherein M: maker；2: the qualification result of bacillus subtilis before inducing；3: the qualification result of bacillus subtilis after induction.

Dichlorodiphenyl Acetate bacillus, bacillus amyloliquefaciens, Bei Laisi bacillus, Bacillus cercus, bacillus pumilus, production Before protein Candida, bacillus licheniformis, yellowish-brown pseudomonad, methanogen, denitrifying bacteria are induced respectively and induce Identification afterwards, the results showed that abductive approach provided by the invention successfully obtains the mixed of above-mentioned bacterial strains symbiosis culture under field conditions (factors) Combined bacteria agent.

Detection primer is as shown in table 4 below:

Table 4

	Sequence (5 ' -3 ')	Sequence number
			Lactobacillus plantarum-F	AGAGTTTGATCCTGGCTCAG	SEQ ID NO.15
Lactobacillus plantarum-R	CTACGGCTACCTTGTTACGA	SEQ ID NO.16

Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

SEQUENCE LISTING

<110>Zhejiang Dai Jun biological medicine Science and Technology Ltd.

<120>the complex microorganism symbiosis Fiber differentiation technology based on SOD and microbe symbiotic culture microbial inoculum and its application

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 20

<212> DNA

<213>artificial sequence

<400> 2

aaggaggtga tccagccgca 20

<210> 3

<211> 18

<212> DNA

<213>artificial sequence

<400> 3

tccggttgat cccgccgg 18

<210> 4

<211> 20

<212> DNA

<213>artificial sequence

<400> 4

ccgtcaattc ctttgagttt 20

<210> 5

<211> 21

<212> DNA

<213>artificial sequence

<400> 5

agagtttgat cctggcttta g 21

<210> 6

<211> 21

<212> DNA

<213>artificial sequence

<400> 6

acggctacct tgttacgact t 21

<210> 7

<211> 20

<212> DNA

<213>artificial sequence

<400> 7

agagtttgat cctggctcag 20

<210> 8

<211> 19

<212> DNA

<213>artificial sequence

<400> 8

ggttaccttg ttacgactt 19

<210> 9

<211> 1368

<212> DNA

<213>denitrifying bacteria

<400> 9

tgcagtcgag cggatgaagg gagcttgctc tctgattcag cggcggacgg gtgagtaatg 60

cctaggaatc tgcctgatag tgggggacaa cgcgaaagga acgctaatac cgcatacgtc 120

ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct aggtcggatt 180

agctagttgg tgaggtaacg gctcaccaag gcgacgatcc gtaactggtc tgagaggatg 240

atcagtcaca ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat 300

attggacaat gggcgaaagc ctgatccagc catgccgcgt gtgtgaagaa ggtcttcgga 360

ttgtaaagca caagttggga ggaagggcat taacctaata cgttagtgtt ttgacgttac 420

cgacgaataa gcaccggcta acttcgtgcc agcagccgcg gtaatacgaa gggtgcaagc 480

gttaatcgga attactgggc gtaaagcgcg cgtaggtggg ttaagttgaa tgtgaaagcc 540

ccgggctcaa cctgggaact gcatccaaaa ctggcaagct agagtatggc agagggtggt 600

ggaacctgtg tagcggtgaa atgcgtagat ataggaagga acaccagtgg cgaaggcgac 660

cacctgggct aatactgaca ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 720

cctggtagtc cacgccgtaa acgatgtcga ctagccgttg ggatccttga gatcttagtg 780

gcgcagctaa cgcattaagt cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa 840

tgaattgacg ggggcccgca caagcggtgg agcatgtgga attcgaagca acgcgaagaa 900

ccttaccagg ccttgacatg cagagaaccc agagatggat tggtgccttc gggaactctg 960

acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta 1020

acgagcgcaa cccttgtcct tagttaccag cacgttaagg tgggcactct aaggagactg 1080

ccggtgacaa accggaggaa ggtggggatg acgtcaagtc atcatggccc ttacggcctg 1140

ggctacacac gtgctacaat ggtcggtaca aagggttgcc aagccgcgag gtggagctaa 1200

tcccataaaa ccgatcgtag tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa 1260

tcgctagtaa tcgtgaatca gaatgtcacg gtgaatacgt tcccgggcct tgtacacacc 1320

gcccgtcaca ccatgggagt gggttgctcc agaagtagct agtctaac 1368

<210> 10

<211> 18

<212> DNA

<213>artificial sequence

<400> 10

agagtttgat cctggcag 18

<210> 11

<211> 19

<212> DNA

<213>artificial sequence

<400> 11

gggtaccttg ttacgactt 19

<210> 12

<211> 20

<212> DNA

<213>artificial sequence

<400> 12

agagtttgat cctggctcag 20

<210> 13

<211> 20

<212> DNA

<213>artificial sequence

<400> 13

ctacggctac cttgttacga 20

<210> 14

<211> 1097

<212> DNA

<213>bacillus licheniformis

<400> 14

tgtcacttcg gcggctggct ccaaaaggtt acctcaccga cttcgggtgt tacaaactct 60

cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc 120

cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat ccgaactgag 180

aacagatttg tgggattggc ttagcctcgc ggcttcgctg ccctttgttc tgcccattgt 240

agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct 300

ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa 360

gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat 420

gcaccacctg tcactctgcc cccgaagggg aagccctatc tctagggttg tcagaggatg 480

tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt 540

gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg 600

cttaatgcgt ttgctgcagc actaaagggc ggaaaccctc taacacttag cactcatcgt 660

ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgcgcctcag 720

cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat 780

ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa 840

tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgcgcg 900

ctttacgccc aataattccc ggaaacgctt gccacctacg tattaccgcg gctgctggca 960

cgtagtttag ccgtggcttt ctgggttagg taccgtcaag gtgccgccct attcgaacgg 1020

tactgtctcc tacacagagt tttacgatcc gaaaaccttc atcactcacg cgggcgttgc 1080

tccgtcagaa ctttcgt 1097

<210> 15

<211> 20

<212> DNA

<213>artificial sequence

<400> 15

agagtttgat cctggctcag 20

<210> 16

<211> 20

<212> DNA

<213>artificial sequence

<400> 16

ctacggctac cttgttacga 20

Claims

1. a kind of abductive approach of microbe symbiotic culture, which comprises the following steps:

First kind bacterium and the second class bacterium by induction processing are mixed, superoxides is added with the first preset interval time Mutase, and, to preset O when incrementss improve culture₂20%-22% is measured, microbe symbiotic culture microbial inoculum is obtained；

The induction processing includes: that superoxide dismutase is added with the first preset interval time in the second class bacterium incubation Enzyme, and, O when predetermined amount improves culture is increased with the second preset interval time₂Measure 5%-20%；

Wherein, the first kind bacterium and the second class bacterium are different.

2. abductive approach according to claim 1, which is characterized in that the training of aerobic bacteria, facultative anaerobic bacteria or mixed culture Feeding base independently includes: 5-10g/L brown sugar, 5-15g/L peptone, 1-5g/L powdered beef and 3-7g/L sodium chloride, pH7.2- 7.4；

Preferably, the culture medium of the anaerobic bacteria includes: 15-25g/L pancreas casein peptone, the thio second of 3-7g/L NaCl, 1-3g/L Alkyd sodium, 0.5-2g/L sodium formaldehyde sulphoxylate, 0.001-0.005g/L methylene blue and 5-10g/L brown sugar, pH7.4-7.6.

3. abductive approach according to claim 1, which is characterized in that first preset interval time is 1-5h；

Preferably, the additional amount of the superoxide dismutase is 0.005%-0.5% (w/v, 20000IU/g), preferably 0.005%-0.1% (w/v, 20000IU/g), further preferably 0.005%-0.05% (w/v, 20000IU/g)；

Preferably, the default incrementss are 1%-8%/day, preferably 1%-5%；

Preferably, second preset interval time is 2-3 days；

Preferably, the predetermined amount is 1%-5%；

It preferably, further include by CO in the abductive approach₂The step of content is reduced to 0.02%-0.5% from 9%-11%.

4. abductive approach according to claim 1, which is characterized in that aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria are independent When ground includes two or more bacterial strains, the bacterial strain is co-cultured 2-3 days, aerobic bacteria, anaerobic bacteria or facultative anaerobic bacteria are obtained.

5. abductive approach according to claim 1, which is characterized in that co-culture system is added in first kind bacterium or the second class bacterium Bacteria concentration independently be 10⁶-10⁸Cfu/ml, inoculum concentration 2-5%.

6. abductive approach according to claim 1-5, which is characterized in that the aerobic bacteria include acetobacter xylinum, At least one of bacillus subtilis, nitrobacteria, acetobacter or nitrogen-fixing bacteria.

7. abductive approach according to claim 1-5, which is characterized in that the anaerobic bacteria include clostridium butyricum, At least one of Bacillus acidi lactici, Bifidobacterium, methanogen or denitrifying bacteria.

8. abductive approach according to claim 1-5, which is characterized in that the facultative anaerobic bacteria includes that goldbeater's skin is bright Beading bacterium, saccharomycete, bacillus amyloliquefaciens, Bei Laisi bacillus, Bacillus cercus, bacillus pumilus, plant cream At least one of bacillus, candida utili, bacillus licheniformis or yellowish-brown pseudomonad.

9. the microbe symbiotic culture microbial inoculum that the described in any item abductive approach of claim 1-8 obtain.

10. the described in any item abductive approach of claim 1-8 or microbe symbiotic culture microbial inoculum as claimed in claim 9 are such as Lower A)-D) application in any one:

A) effective component of animal or plant is extracted；

B microbial-bacterial fertilizer) is prepared；

C) degradation noxious material；

D) degradation larger molecular organics matter.