CN109355196A - A kind of fungi separation method promoting manioc waste substrate fermentation process - Google Patents
A kind of fungi separation method promoting manioc waste substrate fermentation process Download PDFInfo
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- CN109355196A CN109355196A CN201811230079.1A CN201811230079A CN109355196A CN 109355196 A CN109355196 A CN 109355196A CN 201811230079 A CN201811230079 A CN 201811230079A CN 109355196 A CN109355196 A CN 109355196A
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Abstract
The invention discloses a kind of fungi separation methods for promoting manioc waste substrate fermentation process.This method includes establishing fresh manioc waste fermentation heap body, spontaneous fermentation；It is sampled by stages according to the critical stage of spontaneous fermentation process in initial period, temperature raising period, four periods of megathermal period and decomposed phase；Sampling strain is isolated and purified, the fungi for purifying culture is inoculated into Cellulose and congo red differential medium and is cultivated, and the bacterial strain of cellulose-decomposing ability is provided according to the presence or absence of Congo red culture medium transparent circle primary dcreening operation；The strain inoculated just sifted out is fermented secondary screening into manioc waste solid medium, and weight-loss ratio according to solid fermentation manioc waste and content of cellulose filter out the fungi for promoting manioc waste substrate fermentation effect good.Method provided by the invention can rapidly and efficiently excavate out the microorganism beneficial to manioc waste substrate fermentation, to promote the preparation of manioc waste substrate fermentation process microbial bacterial agent to lay the foundation, and provide technical support for the matrixing utilization of manioc waste waste.
Description
Technical field
The invention belongs to substrate microorganism fermentation technical fields, are related to a kind of fungi for promoting manioc waste substrate fermentation process
Separation method.
Background technique
Cassava is the important industrial crops of south China, and manioc waste is as remaining organic solid after cassava processing industry alcohol
Body waste, mainly based on cellulose, hemicellulose and lignin, rich in the beneficial microelement of a variety of pairs of animals and plants.Mesh
Before, fresh manioc waste is used there is also more difficulty, in addition to seldom a part is for animal feed, greatly because its humidity is big
Majority be all it is unprocessed be just directly discharged in environment, not only result in waste of resources, and to ecological environment threaten it is larger.Cause
This, the recycling recycling of manioc waste has become southern region of China solid waste management urgent need to solve the problem.
The matrixing utilization of manioc waste is to solve one of the preferred approach of this problem.Currently, in relation to utilizing the wood after fermentation
Potato slag makes some progress as Vegetable culture medium or as the crop seedlings base Quality Research such as capsicum, cucumber, and
Manioc waste can partially substitute turf, compensate for the problem of turf resource anxiety to a certain extent.But manioc waste substrate fermentation process
Study it is less, especially influence manioc waste course of fermentation microbe research it is less.Therefore, it is badly in need of developing sending out manioc waste matrix
The beneficial microbial bacterial agent of ferment pollutes environment to manioc waste is reduced, and improving solid waste resource recovery and utilizing has important show
Sincere justice.
Summary of the invention
The object of the invention is based on above-mentioned status and the fungi of a kind of promotion manioc waste substrate fermentation process point that provides
From method.This method can provide strong technical foundation for the matrixing utilization of manioc waste.
Technology contents disclosed by the invention are as follows
A kind of fungi separation method promoting manioc waste substrate fermentation process, it is characterised in that including establishing fresh manioc waste
Fermentation heap body, spontaneous fermentation；According to the critical stage of spontaneous fermentation process in initial period, temperature raising period, megathermal period and decomposed phase
Four periods sample by stages；Sampling strain is isolated and purified, the fungi for purifying culture is inoculated into the Congo red culture of cellulose
Base is cultivated, and the bacterial strain of cellulose-decomposing ability is provided according to the presence or absence of Congo red culture medium transparent circle primary dcreening operation；It will be first
The strain inoculated sifted out is fermented secondary screening, and weight-loss ratio and fiber according to solid fermentation manioc waste into manioc waste solid medium
Cellulose content filters out the fungi for promoting manioc waste substrate fermentation effect good.
Preferably, the length of fresh manioc waste fermentation heap body is greater than 2m to one kind as the method for the present invention, and width is greater than
1m is highly greater than 1m, and it is primary to take every two days turning bodies.
One kind as the method for the present invention preferably according to manioc waste course of fermentation, is divided into four critical stage different temperatures
Batch sampling, initial period heap temperature are sampled at 25-27 DEG C, temperature raising period sampling when heap temperature reaches 35-45 DEG C, high temperature
Phase reaches 55-60 DEG C of sampling, the sampling when heap temperature reaches 25-27 DEG C of decomposed phase in heap temperature；It is further preferably initial
Phase sampling when heap temperature reaches 27 DEG C, temperature raising period sampling when heap temperature reaches 35 DEG C and 45 DEG C, the megathermal period is in heap body
Temperature reaches 55 DEG C and 60 DEG C samplings, the sampling when heap temperature reaches 27 DEG C of decomposed phase.
Preferably, described include: respectively by sample to sampling strain and isolate and purify to one kind as the method for the present invention
It is prepared into the suspension of a certain concentration gradient, initial gross separation is then carried out using martin substratum；Again by martin substratum
The fungi of middle growth is inoculated into potato dextrose agar and carries out purifying secondary culture.
As the further preferred of the method for the present invention, the suspension bacteria liquid the preparation method comprises the following steps: weighing fresh sample
10g is put into the conical flask for containing 90mL sterile water, 200 turns of concussion 40min, filtering, filtrate gradient dilutions are made 10-1~10-4
The bacteria suspension of concentration gradient.
As the further preferred of the method for the present invention, the initial gross separation is used and is coated on Martin solid medium
The time of method, culture is 3-5d, and cultivation temperature is consistent with the probe temperature of each sample.
As the further preferred of the method for the present invention, which is characterized in that the purifying secondary culture, which uses, chooses bacterium scribing line
Method, and want continuous purification culture 2-3 times.
One kind as the method for the present invention preferably, the temperature when temperature and sampling of the Cellulose and congo red differential medium culture
Degree is consistent, incubation time 3-5d.
Preferably, the fermentation of manioc waste solid medium is carried out one kind as the method for the present invention in conical flask, at 28 DEG C
Ferment 10d, and primary every three days shaking flasks.
Preferably, the method further includes carrying out bacterium to bacterial strain before secondary screening or after secondary screening to one kind as the method for the present invention
The step of kind identification.
As the further preferred of the method for the present invention, comprising the following steps:
(1) fresh manioc waste fermentation heap body, spontaneous fermentation are established；The length of heap body is greater than 2m, and width is greater than 1m, high
Degree is greater than 1m, and it is primary to take every two days turning bodies；
(2) sampled by stages according to the critical stage of spontaneous fermentation process, respectively initial period, temperature raising period, the megathermal period and
Four periods of decomposed phase；Temperature when sampling is respectively as follows: 27 DEG C of initial period；35 DEG C and 45 DEG C of temperature raising period；55 DEG C of megathermal period and 60
℃；27 DEG C of decomposed phase；
(3) sample that step (2) is taken stage by stage, respectively by sample preparation at 10-1-10-4The suspension of concentration gradient,
It is then applied in martin substratum and cultivates, the time of culture is 3-5d, and cultivation temperature is consistent with the temperature of sample phase；
(4) fungi grown to martin substratum in step (3) is inoculated into potato Portugal using the method for choosing bacterium scribing line
Grape sugar agar medium carries out purifying secondary culture 2-3 times；
(5) it to the fungi of step (4) purifying culture, is inoculated into Cellulose and congo red differential medium and is cultivated, the temperature of culture
Degree is consistent with temperature when sampling, incubation time 3-5d, and is filtered out and have according to the presence or absence of Congo red culture medium transparent circle
The bacterial strain of cellulose-decomposing ability.
(6) the Internal Transcribed Spacer ITS sequencing is carried out to the bacterial strain that step (5) filters out to compare；
(7) ferment effect comparison is carried out to the fungal bacterial strain that step (6) identifies, the fungi of step (6) is inoculated with
Ferment at 28 DEG C 10d into manioc waste solid medium, and primary every three days shaking flasks, and according to solid fermentation manioc waste
Weight-loss ratio and content of cellulose, which filter out, promotes the preferable fungi of manioc waste substrate fermentation effect.
The formula of heretofore described culture medium is preferably as follows:
(1) martin substratum: peptone 5g/L, glucose 10g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.5g/L, chain
Mycin 0.1g/L, rose-bengal 0.033g/L, agar powder 18g/L, tap water 1000mL.
(2) potato dextrose agar: potato 200g/L, glucose 20g/L, agar powder 18g/L, tap water
1000mL。
(3) Congo red culture medium: carboxymethyl cellulose 2.0g/L, ammonium sulfate 2.0g/L, epsom salt 0.5g/L, phosphoric acid
Hydrogen dipotassium 1.0g/L, sodium chloride 0.5g/L, agar powder 18g/L, tap water 1000mL, pH 7.0.
(4) manioc waste solid fermentation culture medium: cassava ground-slag 10g, fermented nutritive liquid 15mL.Fermented nutritive liquid: peptone
10g/L, MgSO4·7H2O 2.5g/L, KH2PO45.0g/L, NaCl 2.5g/L, yeast extract 2.5g/L, CaCl21g/L, water
1000mL, pH 7.0.
The fungi strain that the present invention isolates finds Penicillium bacterium after cultivating 10d in manioc waste solid medium
The weight-loss ratio of manioc waste can be improved, and reduces manioc waste content of cellulose, ferment effect is better than other strains.
The utility model has the advantages that
The present invention provides a kind of fungi separation methods for promoting manioc waste substrate fermentation process, can rapidly and efficiently excavate
The microorganism beneficial to manioc waste substrate fermentation out, realize manioc waste substrate fermentation whole process fungi separation, culture with
Identification provides foundation in the effect of manioc waste substrate fermentation process to fungi, and promotes manioc waste fermentation for further preparation
Microbial inoculum provides technical support.The it is proposed of the method for the present invention, it is matrixing to manioc waste to utilize the scientific meaning and application with reuse
Value.
Detailed description of the invention
Fig. 1: the schematic diagram of the promotion manioc waste fermented fungal separation process of embodiment 1
Fig. 2: manioc waste fermentation critical stage fungi separates situation
Fig. 3: growing state of some fungi in potato dextrose agar
Fig. 4: addition fungi fermentation manioc waste weight-loss ratio analysis
It adds fungi fermentation manioc waste weight-loss ratio and analyzes note: (I -1: Trichoderma harzianum (Trichoderma harzianum),
I -2: Trichoderma viride (Trichoderma viride), I -3: Penicillium (Penicillium sp), I -4: thermophilic pine mould
(Penicillium pinophilum), I -5: A Sasi trichosporon cutaneum (Trichosporon asahii), CK: control (does not add
Add fungi))
Fig. 5: addition fungi fermentation manioc waste content of cellulose analysis
Note: (I -1: Trichoderma harzianum (Trichoderma harzianum), I -2: Trichoderma viride (Trichoderma
Viride), I -3: Penicillium (Penicillium sp), I -4: thermophilic pine mould (Penicillium pinophilum), I -5:
A Sasi trichosporon cutaneum (Trichosporon asahii), CK: control (not adding fungi))
Specific embodiment
Embodiment 1
One, test material
Fresh manioc waste is derived from the Xinghua County source Xin Tu matrix fertilizer Co., Ltd, manioc waste water content 68%,
PH=5.93, EC=4.96mScm-1, total carbon 26.95gkg-1, total phosphorus 9.10gkg-1, total potassium 4.46gkg-1, fiber
Element 35%, hemicellulose 22%, lignin 14%.
Heap body long 2.5m, wide 1.5m, the high 1m of manioc waste substrate fermentation, and plastic covering film, every 3d turning are primary.Horse
Ding Shi culture medium: peptone 5g/L, glucose 10g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.5g/L, streptomysin 0.1g/L, Meng
Add and draws red 0.033g/L, agar powder 18g/L, tap water 1000mL.
Potato dextrose agar: potato 200g/L, glucose 20g/L, agar powder 18g/L, tap water
1000mL。
Congo red culture medium: carboxymethyl cellulose 2.0g/L, ammonium sulfate 2.0g/L, epsom salt 0.5g/L, phosphoric acid hydrogen
Dipotassium 1.0g/L, sodium chloride 0.5g/L, agar powder 18g/L, tap water 1000mL, pH 7.0.
Two, test method
1, the measurement of temperature: using alcohol thermometer, measures respectively in morning 10:00 and 4:00 in afternoon, thermometer is inserted
Measurement at the front, rear, left and right of heap body and 5, center position 30cm.
2, sampling method: according to manioc waste course of fermentation, it is divided into four critical stage different temperatures batch samplings.Initial period
The sampling when heap temperature reaches 27 DEG C, temperature raising period sampling when heap temperature reaches 35 DEG C and 45 DEG C, the megathermal period is in heap body temperature
Degree reaches 55 DEG C and 60 DEG C samplings, and it is sampling that the decomposed phase, which reaches 27 DEG C in heap temperature,.5 point samplings are used when sampling, in heap
It samples, and is fitted into clean plastics at body depth 30cm, tied, marked, in record sampling time etc., be stored in 4 DEG C of refrigerators later
In it is spare.
3, fungi separates
(1) preparation martin substratum, 121 DEG C of high pressure sterilization 20min, inverted plate, culture medium cooling are spare.
(2) bacteria suspension of sample is prepared in batches: being weighed manioc waste sample 10g, is put into the conical flask containing 90mL sterile water
In, it is put into shaking table concussion (temperature setting is consistent with probe temperature, 200 turns of 40min), is then filtered with sterile gauze, at this time
Bacterial concentration is 10 in filtrate-1, 1mL filtrate (bacterial concentration 10 is drawn with sterilized liquid-transfering gun on the super-clean bench-1) be added
It fills and is mixed well in the sterile centrifugation tube of 9mL sterile water, bacterial concentration is 10 at this time-2, and so on be made 10-2~10-4It is several
The suspension bacteria liquid of kind dilution.
(3) it is coated with: in superclean bench, by culture dish label, choosing 10-2、10-3、10-4Three concentration gradients, each
3 repetitions are arranged in concentration gradient, and then each ware is put into the bacteria suspension of the corresponding dilution of 0.1mL, then will with sterile spreading rod
Bacterium solution is spreadable, stands 8min at room temperature.
(4) it cultivates: being inverted culture in biochemical cultivation case, temperature setting is consistent with the temperature marked when sample.
4, fungi purifying culture
Contain streptomysin in martin substratum, bacterium cannot grow with actinomyces, and only fungi can grow.Therefore will
Fungi in martin substratum is purified using the method for scribing line, culture is purified on potato dextrose agar, often
Plant isolated fungi continuous purification 2 times.
5, fungi screening culture
Fungi screening culture, using Cellulose and congo red differential medium screening method, mainly according to Congo red culture medium transparent circle
The presence or absence of judged.
(1) the Congo red culture medium of preparation, 121 DEG C of high pressure sterilization 20min, inverted plate, culture medium cooling are spare.
(2) in super-clean bench, the cake block of fungi is taken with punch, is then seeded into Congo red culture medium, in biochemistry
Culture 6d is inverted in incubator, cultivation temperature setting is consistent with sample temperature.
(3) after cultivating 6 days, 0.3% Congo red solution is added dropwise in the culture dish with bacterium colony, is stood after shaking up
30min is dyed, then rinses Congo red culture medium with 1M NaCl solution, is rinsed 5~6 times back and forth.
(4) according to the presence or absence of transparent circle in Congo red culture medium, 5 kinds of fungies with cellulose-decomposing ability are filtered out.
6, fungi strain is identified
(1) genomic DNA for filtering out fungi is extracted.
(2) to the fungal genomic DNA of said extracted, PCR amplification is carried out to ITS4 and ITS1f using universal primer.
(3) PCR product is sent to biotech firm and is sequenced, sequencing result is subjected to Blast in ncbi database, chooses ratio
Result to similarity 98% or more.The sequencing result of the one plant of Penicillium bacterial strain wherein filtered out such as SEQ ID NO.1 institute
Show.
The present invention isolates as follows with the fungi strain for promoting manioc waste to ferment from manioc waste fermentation critical stage:
(note: " 1 ", which represents, bacterial strain；"-" is represented without bacterial strain)
7, manioc waste fermentation test
(1) manioc waste solid fermentation culture medium is prepared
1) match solid fermentation nutrient solution, peptone 10g/L, MgSO4·7H2O 2.5g/L, KH2PO45.0g/L, NaCl
2.5g/L, yeast extract 2.5g/L, CaCl21g/L, water 1000mL, pH 7.0.
2) 60 meshes are crossed after manioc waste dries pulverizing, weigh 10g in conical flask, and 15mL solid fermentation nutrition is added
Liquid.
3) 121 DEG C of high pressure sterilization 20min of solid medium.
(2) inoculating strain
1) the above 5 kinds of strains isolated are inoculated into respectively in potato dextrose broth, under the conditions of 28 DEG C,
200 turns are shaken bacterium culture 4d.
2) initial spore concentration of each strain is adjusted to 1 × 10 with aqua sterilisa7A/mL.
3) manioc waste solid medium is inoculated with bacterium solution 2mL.
4) and one group of setting is not added microbial inoculum and the control (CK) of sterile water is only added to handle.
5) three repetitions are arranged in each processing.
(3) manioc waste solid-substrate fermentation
Fermentation condition cultivates 10d under the conditions of being 28 DEG C, and primary every 3d shaking flask, keeps its fermentation uniform.
(4) measurement of manioc waste weight-loss ratio and content of cellulose.
No matter from the point of view of weight-loss ratio or content of cellulose, Penicillium is to the ferment effect of manioc waste all than other
Several fungis are good, and wherein the effect of Penicillium is again better than thermophilic loose mould in mould, and weight-loss ratio is than compareing high by 1.3%, cellulose
Content reduces 10.67% than control.
Claims (10)
1. a kind of fungi separation method for promoting manioc waste substrate fermentation process, it is characterised in that including establishing fresh manioc waste hair
Ferment heap body, spontaneous fermentation；According to the critical stage of spontaneous fermentation process in initial period, temperature raising period, megathermal period and decomposed phase four
A period samples by stages；Sampling strain is isolated and purified, the fungi for purifying culture is inoculated into Cellulose and congo red differential medium
It is cultivated, and provides the bacterial strain of cellulose-decomposing ability according to the presence or absence of Congo red culture medium transparent circle primary dcreening operation；By primary dcreening operation
Strain inoculated out is fermented secondary screening, and weight-loss ratio and cellulose according to solid fermentation manioc waste into manioc waste solid medium
Content filters out the fungi for promoting manioc waste substrate fermentation effect good.
2. according to the method described in claim 1, it is characterized in that the length of fresh manioc waste fermentation heap body is greater than 2m, width
It is greater than 1m, is highly greater than 1m, and it is primary to take every two days turning bodies.
3. according to the method described in claim 1, it is characterized in that being divided into four critical stages not according to manioc waste course of fermentation
Synthermal batch sampling, initial period sampling when heap temperature is 25-27 DEG C, temperature raising period is when heap temperature reaches 35-45 DEG C
Sampling, megathermal period reach 55-60 DEG C of sampling, the sampling when heap temperature reaches 25-27 DEG C of decomposed phase in heap temperature；It is preferred that just
The sampling when heap temperature reaches 27 DEG C of phase beginning, temperature raising period sampling when heap temperature reaches 35 DEG C and 45 DEG C, the megathermal period is in heap
Temperature reaches 55 DEG C and 60 DEG C samplings, the sampling when heap temperature reaches 27 DEG C of decomposed phase.
4. according to the method described in claim 1, it is characterized in that described includes: difference to sampling strain and isolate and purify
By sample preparation at the suspension of a certain concentration gradient, initial gross separation is then carried out using martin substratum；Again by Martin
The fungi grown in culture medium is inoculated into potato dextrose agar and carries out purifying secondary culture.
5. according to the method described in claim 4, it is characterized in that the suspension bacteria liquid the preparation method comprises the following steps: weighing fresh
Sample 10g is put into the conical flask for containing 90mL sterile water, 200 turns of concussion 40min, filtering, filtrate gradient dilutions are made 10-1-
10-4The bacteria suspension of concentration gradient.
6. according to the method described in claim 4, it is characterized in that, the initial gross separation uses on Martin solid medium
The time of the method for coating, culture is 3-5d, and cultivation temperature is consistent with the probe temperature of each sample；The purifying secondary culture
Using the method for choosing bacterium scribing line, and want continuous purification culture 2-3 times.
7. the method according to claim 1, wherein the temperature of the Cellulose and congo red differential medium culture with take
Temperature when sample is consistent, incubation time 3-5d.
8. the method according to claim 1, wherein the fermentation of manioc waste solid medium in conical flask into
It goes, ferment 10d at 28 DEG C, and primary every three days shaking flasks.
9. according to the method described in claim 1, it is characterized in that the method further includes before secondary screening or after secondary screening to bacterium
Strain carries out the step of strain idenfication.
10. method according to claim 1 to 9, it is characterised in that the following steps are included:
(1) fresh manioc waste fermentation heap body, spontaneous fermentation are established；The length of heap body is greater than 2m, and width is greater than 1m, highly wants
Greater than 1m, and it is primary to take every two days turning bodies；
(2) it is sampled by stages according to the critical stage of spontaneous fermentation process, respectively initial period, temperature raising period, megathermal period and decomposed
Four periods of phase；Temperature when sampling is respectively as follows: 27 DEG C of initial period；35 DEG C and 45 DEG C of temperature raising period；55 DEG C and 60 DEG C of megathermal period；It is rotten
27 DEG C of ripe phase；
(3) sample that step (2) is taken stage by stage, respectively by sample preparation at 10-1~10-4The suspension of concentration gradient, then
It is applied in martin substratum and cultivates, the time of culture is 3-5d, and cultivation temperature is consistent with the temperature of sample phase；
(4) fungi grown to martin substratum in step (3) is inoculated into potato glucose using the method for choosing bacterium scribing line
Agar medium carries out purifying secondary culture 2-3 times；
(5) to step (4) purifying culture fungi, be inoculated into Cellulose and congo red differential medium and cultivated, the temperature of culture with
Temperature when sampling is consistent, and incubation time is 3~5d, and is filtered out according to the presence or absence of Congo red culture medium transparent circle with fiber
The bacterial strain of plain capacity of decomposition.
(6) the Internal Transcribed Spacer ITS sequencing is carried out to the bacterial strain that step (5) filters out to compare；
(7) ferment effect comparison is carried out to the fungal bacterial strain that step (6) identifies, the fungi of step (6) is inoculated into wood
Ferment at 28 DEG C 10d in potato slag solid medium, and primary every three days shaking flasks, and the weightlessness according to solid fermentation manioc waste
Rate and content of cellulose, which filter out, promotes the preferable fungi of manioc waste substrate fermentation effect.
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| CN110358704A (en) * | 2019-06-26 | 2019-10-22 | 南京农业大学 | A kind of manioc waste fermentation special bacteria agent and preparation method thereof |
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| CN106047730A (en) * | 2016-08-19 | 2016-10-26 | 广西大学 | Application of Penicillium oxalicum EU2101 in preparation of cellulase preparation and degradation of cellulose |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358704A (en) * | 2019-06-26 | 2019-10-22 | 南京农业大学 | A kind of manioc waste fermentation special bacteria agent and preparation method thereof |
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