CN106119144B - A kind of conversion peanut shell hydrolysate prepares the method and bacterial strain of microbial flocculant - Google Patents
A kind of conversion peanut shell hydrolysate prepares the method and bacterial strain of microbial flocculant Download PDFInfo
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- CN106119144B CN106119144B CN201610220636.6A CN201610220636A CN106119144B CN 106119144 B CN106119144 B CN 106119144B CN 201610220636 A CN201610220636 A CN 201610220636A CN 106119144 B CN106119144 B CN 106119144B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
Abstract
The present invention relates to methods and bacterial strain that a kind of conversion peanut shell hydrolysate prepares microbial flocculant, belong to microbial preparation field.Preparation process is as follows: by strainPseudomonas sp.BWL918 is inoculated into seed liquid culture medium, and 28 DEG C of shaking table cultures prepare seed liquor；Seed liquor is transferred in 1% ratio using peanut shell hydrolysate as in the fermentation medium of carbon source, 28 DEG C, 180 rpm shaking table culture, 48 h；By fermentation liquid at 4 DEG C, after 10000 rpm are centrifuged 10 min, collecting supernatant is liquid microbe flocculant；The dehydrated alcohol of 2 times of volumes pre-cooling is added into liquid fermentation liquid, sediment is collected by centrifugation and freeze-drying obtains sterling microbial flocculant.The present invention can use peanut shell hydrolysate as the carbon source of fermentation medium and produce microbial flocculant, can reduce the production cost of microbial flocculant, adapt to large-scale production and industrial applications.
Description
Technical field
The present invention relates to methods and bacterial strain that a kind of conversion peanut shell hydrolysate prepares microbial flocculant, and in particular to benefit
WithPseudomonas The method of sp.BWL918 conversion peanut shell hydrolysate fermenting and producing microbial flocculant.
Background technique
China is large agricultural country, and about 700,000,000 tons of agricultural wastes, such as corn stalk can be generated in annual agricultural production process
Burning processing, but this not only waste of resource, but also pollution ring is usually taken in stalk, peanut shell, soil dynamic test and rice husk etc., people
Border.Cellulose substances rich in these residues, can be converted to carbohydrate, so that other high value added products are produced,
How value product effectively is converted with important economic significance by these cellulose family agricultural wastes.
Flocculant is a kind of substance that can assemble suspended particulate in sedimentation solution, is broadly divided into inorganic flocculating agent (such as sulphur
Sour aluminium, bodied ferric sulfate etc.), organic polymer coargulator (such as polyethyleneimine, polyacrylamide) and natural polymer wadding
Solidifying agent (such as modified starch, chitosan, sodium alginate, chitin and microbial flocculant).Inorganic flocculating agent is in China market
On account for important share, have the demand of hundreds of thousands ton every year, mainly with molysite, aluminium salt is representative.Molysite has corrosivity, is easy
Residual is formed, treated, and water has color, causes secondary pollution；Found in hygiene aluminium salt the intracorporal accumulation of people with it is current
Senile dementia has direct relationship.Organic polymer coargulator is answered extensively due to its good flocculating effect and low cost
For in actual production, using polyacrylamide as representative, but its to be difficult to degrade, monomeric acrylamide is a kind of strong carcinogen, and
With strong neurotoxicity, the acrylamide monomer remained in environment easily causes secondary pollution, the environmentally safe and mankind
Health causes to seriously affect.The natural macromolecule flocculating agents such as chitosan, sodium alginate, chitin are although nontoxic, can safe disposal,
But its flocculation activity is weaker, higher cost, limits its extensive use；Microbial flocculant is novel third generation flocculant,
It is the metabolite generated by microorganism, such as glycoprotein, mucopolysaccharide, protein, cellulose, there is nontoxic, safety
The features such as height, biology is degradable and without secondary pollution, therefore microbial flocculant can be widely used in sewage treatment, especially
It is that there is unique advantage in the techniques such as the post-processing of food and fermentation industry, research is paid more and more attention.
The bacterium for producing flocculant of microbe strain registered at present has tens kinds, such as Bacillus licheniformis X14
Produced microbial flocculant is mainly made of neutral sugar and protein, and Optimal compositions of fermentation medium is 20 g/L of glucose, ferment
Female powder 0.5g/L, urea 0.5g/L；Bacillus firmusS-14 can generate a kind of polysaccharide biological flocculant, best to send out
Ferment culture medium is 10 g/L of glucose, yeast powder 0.5g/L；BacillusSp. the produced biological flocculant of F19 is mainly in
Property the composition such as sugar, uronic acid, amino sugar and protein, Optimal compositions of fermentation medium is 20 g/L of sucrose, 2.5 g/L of yeast powder.
These strain used mediums mostly using expensive carbohydrate as main component, cause microbial flocculant high production cost, limit
The industrial applications of microbial flocculant.
In order to reduce the production cost of microbial flocculant, Guo etc. is using corn stover hydrolysate as bacterial strainRhodococcuserythropolisFermenting carbon source produce microbial flocculant (Bioresource Technology.
2015;177:393-397)；Wang etc. is using rice husk hydrolysate as bacterial strainOchrobactiumciceriThe fermentation carbon of W2
Source produces microbial flocculant (Bioresource technology, 2013,145:259-263)；Chin-Hang Shu
Et al. also using rice husk hydrolysate asSchizophyllum communeCulture medium produce microbial flocculant
(Journal of the Taiwan Institute of Chemical Engineers, 2011, 42(3): 387-
393).But it is had no at present using peanut shell hydrolysate as the research of fermentation medium production microbial flocculant.China is annual
It about more than 1,500 ten thousand tons of peanut yield, ranks first in the world, the annual peanut shell waste for generating about 5,000,000 tons, these peanut shells
Waste is commonly used as fuel, results in waste of resources and environmental pollution.The objects such as cellulose rich in, starch in peanut shell
Matter has the potentiality for developing other high value added products.Bacterial strain uses therefor of the present invention separates the pseudomonad of acquisition for usPseudomonas Sp.BWL918, the bacterial strain can utilize peanut shell hydrolysate fermenting and producing microbial flocculant, can be effectively
The production cost for reducing microbial flocculant, turns waste into wealth, realizes the resource utilization of peanut shell.
Summary of the invention
The present invention provides one kindPseudomonas Sp.BWL918 bacterial strain and its in neutral conditions conversion peanut shell water
The method for solving object production high-activity microorganism flocculant.The bacterial strain has the speed of growth fast, and fermentation period is short, and flocculant yield is high
The advantages that, the production cost of microbial flocculant can be effectively reduced.
The present invention includes the following contents: a kind of conversion peanut shell hydrolysate prepares the bacterial strain of microbial flocculantPseudomonas Sp.BWL918, is preserved in China General Microbiological culture presevation administrative center, and preservation address is court, Beijing
The institute 3 of positive area's North Star West Road 1, culture presevation number are CGMCC 11994, preservation date 2016.01.12.
It is a kind of to utilize bacterial strainPseudomonas Sp.BWL918 conversion peanut shell hydrolysate prepares the side of microbial flocculant
The processing step of method, this method is as follows:
Step 1 crushes peanut shell, and is sieved with 60 mesh mesh screens, and peanut shell powder is added 1.7% by the concentration of 100g/L
(W/W) H2SO4In solution, in 121 DEG C of 120 min of hydrolysis, after cooling, 10 min is centrifuged, supernatant are collected, with Ca (OH)2It adjusts
PH to 7.0 is saved, is centrifuged 10 min again, collects the carbon source that supernatant obtains peanut shell hydrolysate for subsequent fermentation culture medium；
Strain is inoculated into seed liquid culture medium by step 2,28 DEG C of 12 h of shaking table culture, prepares seed liquor；
Seed liquor is transferred in using peanut shell hydrolysate as the fermentation medium of carbon source by step 3 in 1% ratio, is shaken
48 h of bed culture；
Step 4, by fermentation medium at 4 DEG C, be centrifuged 10 min, collect supernatant be liquid microbe flocculant；
Step 5, the dehydrated alcohol that the pre-cooling of 2 times of volumes is added into liquid microbe flocculant, are collected by centrifugation sediment, and
Freeze-drying obtains microbial flocculant product.
Further, seed liquid culture medium described in step 2 is 1 g/L of glucose, 1 g/L of soluble starch, yeast
1 g/L of powder, 1 g/L of tryptone, 1 g/L of acid hydrolyzed casein, 0.1 g/L of epsom salt, 0.6 g/L of dipotassium hydrogen phosphate,
The cultivation temperature of seed liquor is 28 DEG C.
Further, the shaking table culture condition in step 3 is 28 DEG C, 180 rpm shaking table culture, 48 h.
Further, fermentation medium described in step 3 is peanut shell hydrolysate 300mL/L, yeast powder 3 g/L, seven
0.1 g/L of water magnesium sulfate, 0.6 g/L of dipotassium hydrogen phosphate, the initial pH of fermentation liquid are 7.0.
Further, the centrifugal rotational speed of step 1 and step 4 is 10000 rpm.
Further, in the step 5,2 times of volumes pre-coolings are added into the liquid microbe flocculant of step 4 acquisition
Dehydrated alcohol, 10000 rpm be centrifuged 10 min collect precipitating, 70% ethanol washing precipitating after, freeze-drying obtain microorganism wadding
Solidifying agent sterling.
Compared with prior art, the present invention having the advantage that present invention firstly provides produce using peanut shell hydrolysate
Microbial flocculant may be implemented to reduce environmental pollution to the resource utilization of peanut shell.Bacterial strain involved in the present inventionPseudomonas The sp.BWL918 speed of growth is fast, and fermentation period is short, and microbial flocculation yield is high, before having application well
Scape.
Specific embodiment
A kind of conversion peanut shell hydrolysate prepares the bacterial strain of microbial flocculantPseudomonas Sp.BWL918, preservation
In China General Microbiological culture presevation administrative center, culture presevation number is CGMCC 11994.
A kind of processing step that conversion peanut shell hydrolysate prepares microbial flocculant is as follows:
Step 1, by strainPseudomonas Sp. BWL918 is inoculated into seed liquid culture medium, and 28 DEG C of shaking flasks are trained overnight
Support preparation seed liquor；
Seed liquor is transferred in 1% ratio using peanut shell hydrolysate as in the fermentation medium of carbon source, 28 DEG C by step 2,
180 rpm shaking table culture, 48 h；
Step 3, by fermentation medium at 4 DEG C, 10000 rpm are centrifuged 10 min, removal precipitating, and supernatant is that liquid is raw
Object flocculant；
Step 4, the dehydrated alcohol that the pre-cooling of 2 times of volumes is added into liquid fermentation liquid are collected by centrifugation sediment and freeze dry
It is dry to obtain sterling biological flocculant.
In the step 1, strain used is pseudomonadPseudomonas Sp.BWL918, patent culture presevation number
For CGMCC 11994, microbial flocculant can be produced using peanut shell hydrolysate；
In the step 1, seed liquor medium component is as follows: 1 g/L of glucose, 1 g/L of soluble starch, yeast powder
1 g/L, 1 g/L of tryptone, 1 g/L of acid hydrolyzed casein, 0.1 g/L of epsom salt, 0.6 g/L of dipotassium hydrogen phosphate；
In the step 2, the preparation process of peanut shell hydrolysate is as follows: peanut shell being crushed with pulverizer, with 60 mesh
Mesh screen sieving, 1.7%(W/W is added in peanut shell powder by the concentration of 100g/L) H2SO4In solution, 121 DEG C of 120 min of hydrolysis,
After cooling, supernatant is collected by centrifugation, with Ca (OH)2PH to 7 is adjusted, supernatant is collected by centrifugation again and obtains peanut shell hydrolysate；
In the step 2, the ingredient of fermentation medium is as follows: 300 mL/L of peanut shell hydrolysate, yeast powder are nitrogen source 3
G/L, 0.1 g/L of epsom salt, 0.6 g/L of dipotassium hydrogen phosphate.
Specific embodiment 1: microbial flocculant preparation method, the specific steps are as follows:
1, gradient dilution method separation screening function stem, the screening and culturing based component strain separating and identification: are utilized
Are as follows: 0.5 g/L of glucose, 0.5 g/L of soluble starch, 0.5 g/L of yeast powder, 0.5 g/L of tryptone, acid hydrolyzed casein
0.5 g/L, epsom salt 0.05g/L, dipotassium hydrogen phosphate 0.3g/L.After strain separating obtains, purified by four zoning collimation methods
Method obtains pure strain, through 16S rRNA sequence analyze determine the present invention used in strain be pseudomonad (Pseudomonas
Sp.), and it is named asPseudomonas Sp. BWL918, patent culture presevation number are CGMCC 11994。
2, prepared by seed liquor: by pseudomonadPseudomonas Sp.BWL918 is seeded in seed liquid culture medium, seed
Liquid culture medium component is as follows: 1 g/L of glucose, 1 g/L of soluble starch, 1 g/L of yeast powder, 1 g/L of tryptone, sour water solution
1 g/L of casein, 0.1 g/L of epsom salt, 0.6 g/L of dipotassium hydrogen phosphate.Then 28 DEG C, 180 rpm shaking tables are incubated overnight.
3, fermentation prepares microbial flocculant: the seed liquor that step 2 obtains is seeded in fermentation medium in 1% ratio,
Fermentation medium component is as follows: peanut shell powder hydrolysate 300mL/L;3 g/L of yeast powder, 0.1 g/L of epsom salt, phosphoric acid
0.6 g/L of hydrogen dipotassium；Then 28 DEG C, 180rpm shaker fermentation culture 48h.
4, microbial flocculant extracts: the fermentation liquid that step 3 obtains being centrifuged 10 min under the conditions of 10000 rpm, is received
Collect supernatant；The dehydrated alcohol of 2 times of volumes pre-cooling is added into supernatant, 10000 rpm are centrifuged 10 min and collect precipitating, use
After 70% ethanol washing precipitating, freeze-drying obtains microbial flocculant sterling.
5, flocculation activity measures: 100 μ L fermentation liquids are added into 60 mL5 g/L kaolinite soil suspensions；Quickly stirring 2
Then min is slowly stirred 1 min, 1 min of standing sedimentation, take supernatant with 722 type spectrophotometric determination OD550Value, to add
Enter 100 μ L distilled water as control, indicates flocculation activity by calculating flocculating rate.Flocculating rate=(A-B)/A × 100%, A are represented
The OD of supernatant when adding distilled water550Value, B represent the OD of supernatant when adding fermentation liquid550Value.Microbial flocculant pair obtained
Aqueous suspension ofkaolin has apparent flocculating effect.
Claims (7)
1. a kind of pseudomonad strain (Pseudomonas sp.BWL918), is preserved in China General Microbiological culture presevation pipe
Reason center, culture presevation number are CGMCC 11994.
2. the method for preparing microbial flocculant using the conversion peanut shell hydrolysate of pseudomonad described in claim 1, this method
Processing step it is as follows:
Step 1 crushes peanut shell, and is sieved with 60 mesh mesh screens, and 1.7%W/W is added in peanut shell powder by the concentration of 100g/L
H2SO4In solution, in 121 DEG C of hydrolysis 120min, after cooling, it is centrifuged 10min, supernatant is collected, with Ca (OH)2Adjust pH to
7.0, it is centrifuged 10min again, collects the carbon source that supernatant obtains peanut shell hydrolysate for subsequent fermentation culture medium；
Pseudomonad strain described in claim 1 is inoculated into seed liquid culture medium by step 2,28 DEG C of shaking table culture 12h systems
Standby seed liquor；
Seed liquor is transferred in using peanut shell hydrolysate as the fermentation medium of carbon source, shaking table by step 3 in 1% ratio
Cultivate 48h；
Step 4, by fermentation medium at 4 DEG C, be centrifuged 10min, collect supernatant be liquid microbe flocculant；
Step 5, the dehydrated alcohol that the pre-cooling of 2 times of volumes is added into liquid microbe flocculant, are collected by centrifugation sediment, and freeze
It is dried to obtain microbial flocculant product.
3. according to claim 2 prepare microorganism using the conversion peanut shell hydrolysate of pseudomonad described in claim 1
The method of flocculant, which is characterized in that seed liquid culture medium described in step 2 be glucose 1g/L, soluble starch 1g/L,
Yeast powder 1g/L, tryptone 1g/L, acid hydrolyzed casein 1g/L, epsom salt 0.1g/L, dipotassium hydrogen phosphate 0.6g/L, kind
The cultivation temperature of sub- liquid is 28 DEG C.
4. according to claim 2 prepare micro- life using the conversion peanut shell hydrolysate of pseudomonad described in claim 1
The method of object flocculant, which is characterized in that the shaking table culture condition in step 3 is 28 DEG C, 180rpm shaking table culture 48h.
5. according to claim 2 prepare microorganism using the conversion peanut shell hydrolysate of pseudomonad described in claim 1
The method of flocculant, which is characterized in that fermentation medium described in step 3 is peanut shell hydrolysate 300mL/L, yeast powder
3g/L, epsom salt 0.1g/L, dipotassium hydrogen phosphate 0.6g/L, the initial pH of fermentation liquid are 7.0.
6. according to claim 2 prepare microorganism using the conversion peanut shell hydrolysate of pseudomonad described in claim 1
The method of flocculant, which is characterized in that the centrifugal rotational speed of step 1 and step 4 is 10000rpm.
7. according to claim 2 prepare microorganism using the conversion peanut shell hydrolysate of pseudomonad described in claim 1
The method of flocculant, which is characterized in that in the step 5,2 times are added into the liquid microbe flocculant of step 4 acquisition
The dehydrated alcohol of volume pre-cooling, 10000rpm is centrifuged 10min and collects precipitating, and after 70% ethanol washing precipitates, freeze-drying is obtained
Microbial flocculant sterling.
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| CN107815548A (en) * | 2017-11-09 | 2018-03-20 | 合山锌业科技有限公司 | The method that zinc cadmium waste residue produces feed grade monohydrate zinc sulphate and cadmium |
| CN113173646B (en) * | 2021-01-11 | 2022-07-05 | 武夷学院 | Microbial flocculant and preparation method and application thereof |
| CN115232767B (en) * | 2022-07-19 | 2023-04-07 | 山东大湖新材料有限公司 | Method for preparing bioflocculant by Parasphingorabhubus sp |
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