CN108841765A - Steady bacillus and its application in conversion sweet potato stalk production biological flocculant - Google Patents
Steady bacillus and its application in conversion sweet potato stalk production biological flocculant Download PDFInfo
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Abstract
One plant of steady bacillus Empedobacter falsenii BWL1071, deposit number are：CGMCC No.13986.Application of the above-mentioned steady bacillus in conversion sweet potato stalk hydrolysate production biological flocculant, including：Steady bacillus is inoculated in seed liquid culture medium and prepares seed fermentation liquid；Seed fermentation liquid is forwarded to culture in the fermentation liquid culture medium using sweet potato stalk hydrolysate as carbon source and obtains fermentation culture；Supernatant is collected in fermentation culture heating, centrifugation；Dehydrated alcohol is added into supernatant, sediment, pre-cooled ethanol washing is collected by centrifugation, vacuum freezedrying obtains solid biologic flocculant product.Bacterial strain Empedobacter falsenii BWL1071 of the invention can convert sweet potato stalk hydrolysate production biological flocculant, not only improving reduces biological flocculant production cost, the resource utilization of sweet potato stalk may be implemented again, the biological flocculant energy efficient flocculating of generation handles mining wastewater.
Description
Technical field
The invention belongs to field of biotechnology, are related to microorganism and its metabolite, and in particular to one plant of steady bacillus
Empedobacter falsenii BWL1071 and its answering in conversion sweet potato stalk hydrolysate fermenting and producing biological flocculant
With.
Background technique
With the rapid development of our country's economy, environmental problem becomes increasingly conspicuous, and wherein water pollution is most challenging environment
One of problem.A large amount of mining wastewater can be generated in mining site exploitation and process, including mining process draining, tailing pit are overflow
Flowing water and mine drainage etc. mainly contain solid suspension, heavy metal ion and organic pollutant etc., and concentration is much higher than country
The content of discharge standard, especially solid suspension severely exceeds.It is outstanding that flocculant is that one kind can assemble solid in sedimentation solution
The substance of floating particles is widely used in the fields such as sewage treatment.It is broadly divided into three classes：Inorganic flocculating agent, organic polymer
Flocculant and biological flocculant.Flocculant can remove solid suspension and heavy metal ion in mining wastewater with efficient flocculating,
In the recycling of mining wastewater, the fields such as ecological environmental protection are with good application prospect.
Biological flocculant is the polysaccharide and the macromolecular substances such as albumen generated by microbial metabolism, have it is nontoxic, can
Biodegrade, it is without secondary pollution the advantages that, biological flocculant can substitute traditional inorganic flocculating agent and organic polymer flocculation
Agent, reduces environmental pollution and the destruction to the ecosystem.However the production of biological flocculant needs hair of the pure sugar as microorganism
Ferment culture medium, the cost is relatively high for fermenting and producing, limits large-scale industrial production and the application of biological flocculant.In order to drop
The production cost of low biological flocculant, scientific research personnel have carried out a series of research work, and main includes screening superior strain, utilize
Genetic engineering breeding technology improves the yield of existing bacterial strain, and the zymotechnique of optimization biological flocculant production bacterial strain widens biology
Several aspects such as the application field of flocculant.Exploring and finding cheap substitutive medium is also that biological flocculant is effectively reduced to be produced into
This effective way, such as Liu Wei outstanding person et al. are using the hot acid hydrolysate of peanut shell as strain Pseudomonas veronii
L918 fermentation medium carbon source (Bioresource technology, 2016,218:318-325), Guo etc. utilizes rice husk
Carbon source (Bioresource of the hot acid hydrolysate as bacterial strain Rhodococcus erythropolis fermentation medium
technology,2015,177:393-397), Chen etc. is using wastewater containing phenol as bacterial strain Stenotrophomonas
The fermentation medium of maltophilia produces biological flocculant, reduces the production cost of biological flocculant, promotes biological wadding
Solidifying agent is in sewage treatment, the application in the fields such as activated sludge dehydration and microalgae cell harvesting.
China is that maximum sweet potato producing country, every annual planting area are more than 80,000,000 mu in the world.In sweet potato planting process
The middle a large amount of sweet potato straw refuses that can be generated, these sweet potato straw refuses are used as animal and fowl fodder or are simply discarded, make
At the waste of serious biomass resource and environmental pollution.Protein and lignocellulosic substance rich in sweet potato stalk,
It can be decomposed into monosaccharide under the conditions of hot acid, and then be converted into other high value added products.
Summary of the invention
The present invention provides one plant of steady bacillus Empedobacter falsenii BWL1071, deposit number is：
CGMCC No.13986, this constitutes the first aspect of the invention.
The present invention also provides above-mentioned steady bacillus to produce the application in biological flocculant in conversion sweet potato stalk hydrolysate, this
The second aspect of the invention is constituted, is specifically included：
Steady bacillus described in claim 1 is inoculated in seed liquid culture medium by step 1, in 150-200rpm, 29-
Shaking table shake culture 10-14h under conditions of 31 DEG C, prepares seed fermentation liquid；
Seed fermentation liquid obtained in step 1 is forwarded in 1% ratio with sweet potato stalk hydrolysate work by step 2
For in the fermentation liquid culture medium of carbon source, under conditions of 170-190rpm, 33-35 DEG C, shaking table shake culture 23-25h is sent out
Ferment culture solution；
The fermentation culture that step 2 obtains is heated 1h in 80 DEG C of thermostat water baths by step 3, then in 10000rpm,
It is centrifuged 10min under conditions of 4 DEG C, collects supernatant；
The dehydrated alcohol of 2 times of volumes pre-cooling is added in step 4, the supernatant obtained into step 3, precipitating is collected by centrifugation
Sediment is dissolved in a small amount of water, vacuum freezedrying by object with 75% ethanol washing sediment 2 times of pre-cooling, obtains solid
Biological flocculant product.
Further, the constituent of the seed liquid culture medium is as follows：Glucose 5g/L, yeast powder 20g/L, seven water sulphur
Sour magnesium 0.5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, the condition of culture of seed liquor are 170-
190rpm, 29-31 DEG C are incubated overnight.
Further, the constituent of the fermentation liquid culture medium is：Sweet potato stalk hydrolysate 300mL/L, yeast powder
20g/L, epsom salt 0.5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, fermentation condition are
170-190rpm, 33-35 DEG C.
Further, the preparation process of the sweet potato stalk hydrolysate is：By sweet potato stalk and 1.7% sulfuric acid solution by solid
Liquor ratio 1:After 10 ratio mixing, sour water solution 2h in 121 DEG C of high-pressure sterilizing pots；After cooling, mixture is centrifuged through 10000rpm
After 10min, supernatant is collected, is added Ca (OH)2PH to 7.0 is adjusted, then collects supernatant through 10000rpm centrifugation 10min again
Liquid obtains sweet potato stalk hydrolysate.
Beneficial effects of the present invention：
1. bacterial strain Empedobacter falsenii BWL1071 of the invention can convert the production of sweet potato stalk hydrolysate
Biological flocculant, not only improving reduces biological flocculant production cost, and the resource utilization of sweet potato stalk may be implemented；
2. the biological flocculant energy efficient flocculating that bacterial strain Empedobacter falsenii BWL1071 of the invention is generated
Handle mining wastewater.
The culture presevation date of the invention is on 04 5th, 2017, and deposit number is：CGMCC No.13986.Systematic name
Referred to as：Steady bacillus (Empedobacter falsenii) BWL1071, the entitled China General Microbiological strain of depositary institution are protected
Administrative center is hidden, address is No. 3 Institute of Microorganism, Academia Sinica of city of BeiJing, China Chaoyang District North Star West Road 1 institute, postcode
100101。
Specific embodiment：
Embodiment 1：Screening process
The process of pure bacterial strain from drawing materials to filtering out
(1) iron rust sample is acquired from Xuzhou City of Jiangsu Province factory of brass hill area, 1g iron rust sample is suspended in sterile physiological
In salt water, it by sample physiological saline gradient dilution and is successively coated on screening and culturing medium (beef extract 5g/L, sodium chloride 5g/
L, peptone 10g/L, agar powder 20g/L) on.
(2) after coating to plate, culture is inverted in 30 DEG C of constant incubators.Every checking strain growth situation for 24 hours.
According to the different characteristics such as shape, size, color, choosing colony to LB liquid medium (yeast powder 5g/L, NaCl 10g/L, egg
White peptone 10g/L) in, 30 DEG C of constant-temperature tables are placed in, shaking flask culture, condition of culture are carried out：180rpm, culture is for 24 hours.Shaking table terminates
Flocculation activity measurement is carried out to fermentation liquid afterwards, obtains the high bacterial strain BWL1071 of flocculation activity.
Embodiment 2：Strain idenfication
Colonial morphology：Bacterium colony is creamy white, semi-moist, and edge is irregular.
The kind tree status that bacterial strain BWL1081 is determined using 16S rRNA sequence, using bacterial genomes DNA extraction kit
Extract total DNA.The forward primer of PCR clone is 5 '-GAG AGT TTG ATC CTG GCT CAG-3 ', reverse primer 5 '-
CTA CGG CTA CCT TGT TAC GA-3'.It is 50 μ L that PCR, which reacts total volume, in 0.2mL Eppendorf pipe under
Column sequence is added：premix 25μL；2 μ L of forward primer；2 μ L of reverse primer；1 μ L of template DNA；20 μ L of sterile water.PCR condition
For 95 DEG C of initial denaturations 1min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, totally 30 are recycled, 72 DEG C of extensions
10min.The 16S rRNA product obtained after amplification send to sequencing company after the detection of 1% agarose gel electrophoresis and is sequenced, sequencing
As a result as shown in SEQ ID No.1.The 16S rRNA sequence measured is subjected to BLAST comparison in NCBI, finds similarity most
High known array finally found that the similarity highest of bacterial strain BWL1081 Yu Empedobacter falsenii.
Embodiment 3：
The method that bacterial strain Empedobacter falsenii BWL1071 converts sweet potato stalk production biological flocculant, tool
Body embodiment is as follows：
Strain Empedobacter falsenii BWL1071 is inoculated in seed liquid culture medium, 150- by step 1
200rpm, 29-31 DEG C of shaking table culture 10-14h prepare seed fermentation liquid；
Seed liquor described in step 1 is transferred in 1% ratio using sweet potato straw powder as sole carbon source by step 2
In fermentation liquid culture medium, 170-190rpm, 33-35 DEG C of constant-temperature table culture 23-25h；
Fermentation liquid is heated 1h in 80 DEG C of water-baths by step 3, is then centrifuged 10min under conditions of 10000rpm, 4 DEG C,
Collect supernatant；
The dehydrated alcohol of 2 times of volumes pre-cooling is added in step 4, the supernatant obtained into step 3, precipitating is collected by centrifugation
Object is dissolved in a small amount of water, vacuum freeze drying with 75% ethanol washing 2 times, obtains solid biologic flocculant product.
Seed liquid culture medium described in step 1 is glucose 5g/L, yeast powder 20g/L, epsom salt 0.5g/L, phosphorus
Sour hydrogen dipotassium 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, the condition of culture of seed liquor are 170-190rpm, 29-31 DEG C
It is incubated overnight；
Fermentation medium described in step 2 is sweet potato stalk hydrolysate 300mL/L, yeast powder 20g/L, epsom salt
0.5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, fermentation condition 170-190rpm, 33-35
℃。
The preparation method of the hydrolysate of sweet potato stalk described in step 2 is as follows：Sweet potato stalk and 1.7% sulfuric acid solution press solid-liquid
Than 1:After 10 ratio mixing, sour water solution 2h in 121 DEG C of high-pressure sterilizing pots；After cooling, mixture is centrifuged through 10000rpm
After 10min, supernatant is collected, is added Ca (OH)2PH to 7.0 is adjusted, then collects supernatant through 10000rpm centrifugation 10min again
Liquid, to obtain sweet potato stalk hydrolysate.
Embodiment 4
The biological flocculant flocculation treatment mining wastewater that bacterial strain Empedobacter falsenii BWL1071 is generated, tool
Body implementation steps are as follows：
60mL mining wastewater is taken, 100uL bacterial strain Empedobacter falsenii BWL1071 is added into mining wastewater
Fermentation liquid；Quickly stirring 2min, is then slowly stirred 1min, standing sedimentation 1min takes supernatant to use, and uses spectrophotometer
Measure OD550Value indicates biological flocculant to mining wastewater 100 μ L distilled water are added as control by calculating flocculating rate
Flocculation activity.Flocculating rate=(A-B)/A × 100%, A represent the OD of supernatant when adding distilled water550Value, when B representative adds fermentation liquid
The OD of supernatant550Value.The flocculant of bacterial strain Empedobacter falsenii BWL1071 generation is measured to mining wastewater
Application effect, the results showed that biological flocculant obtained is obvious to the flocculating effect of mining wastewater, flocculating rate reach 90% with
On.
Sequence table
<110>Jiangsu Normal University
<120>Steady bacillus and its application in conversion sweet potato stalk production biological flocculant
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1343
<212> DNA
<213>Steady bacillus (Empedobacter falsenii)
<400> 1
agttctttcg ggaactagag accggcgcac gggtgcgtaa cgcgtatgca acttgcccta 60
ctgaaaagga tagcccttcg aaaggaggat taatacttta taacagattt aatggcatca 120
ttagattttg aaagatttat cgcagtagga taggcatgcg taagattagt tagttggtga 180
ggtaacggct caccaagacg atgatcttta gggggcctga gagggtgaac ccccacactg 240
gtactgagac acggaccaga ctcctacggg aggcagcagt gaggaatatt ggacaatggg 300
tggaagcctg atccagccat cccgcgtgta ggatgacggc cttatgggtt gtaaactact 360
tttatctggg gataaaccta cttacgtgta agtagctgaa ggtaccagaa gaataagcac 420
cggctaactc cgtgccagca gccgcggtaa tacggagggt gcaagcgtta tccggattta 480
ttgggtttaa agggtccgta ggcggattaa tcagtcagtg gtgaaatctc atagcttaac 540
tatgaaactg ccattgatac tgttagtctt gagtgatgtt gaagttgctg gaatgtgtag 600
tgtagcggtg aaatgcttag atattacgca gaacaccaat tgcgaaggca ggtgactaaa 660
cattaactga cgctgatgga cgaaagcgtg gggagcgaac aggattagat accctggtag 720
tccacgccgt aaacgatgga tacttgctgt tggattttcg gattcagtgg ctaagcgaaa 780
gttataagta tcccacctgg ggagtacgtt cgcaagaatg aaactcaaag gaattgacgg 840
gggcccgcac aagcggtgga gcatgtggtt taattcgatg atacgcgagg aaccttacca 900
aggcttaaat gcaatttgac agaactagaa atagtttttt cttcggacag aatgcaaggt 960
gctgcatggc tgtcgtcagc tcgtgccgtg aggtgttagg ttaagtcctg caacgagcgc 1020
aacccctatc attagttgcc agcgtttaaa gacggggact ctaatgagac tgccggtgca 1080
aaccgcgagg aaggtgggga cgacgtcaag tcatcacggc ccttacgtct tgggctacac 1140
acgtgctaca atggtaagta cagagggcag ctacttggca acaagatgcg aatctcaaaa 1200
acttatctca gttcggattg gagtctgcaa ctcgactcta tgaagctgga atcgctagta 1260
atcgcatatc agccatgatg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320
agccatggaa gctgggggta cct 1343
Claims (6)
1. one plant of steady bacillus (Empedobacter falsenii.) bacterial strain, deposit number are：CGMCC No.13986.
2. application of the steady bacillus described in claim 1 in conversion sweet potato stalk production biological flocculant.
3. application of the steady bacillus according to claim 2 in conversion sweet potato stalk production biological flocculant, feature exist
In the application specifically includes：
Steady bacillus described in claim 1 is inoculated in seed liquid culture medium by step 1, in 150-200rpm, 29-31 DEG C
Under conditions of shaking table shake culture 10-14h, prepare seed fermentation liquid；
Seed fermentation liquid obtained in step 1 is forwarded in 1% ratio using sweet potato stalk hydrolysate as carbon by step 2
In the fermentation liquid culture medium in source, under conditions of 170-190rpm, 33-35 DEG C, shaking table shake culture 23-25h obtains fermentation training
Nutrient solution；
The fermentation culture that step 2 obtains is heated 1h in 80 DEG C of thermostat water baths by step 3, then in 10000rpm, 4 DEG C
Under conditions of be centrifuged 10min, collect supernatant；
The dehydrated alcohol of 2 times of volumes pre-cooling is added in step 4, the supernatant obtained into step 3, sediment is collected by centrifugation, uses
75% ethanol washing sediment 2 times of pre-cooling, sediment are dissolved in a small amount of water, vacuum freezedrying, obtain solid biologic
Flocculant product.
4. application of the steady bacillus in conversion sweet potato stalk production biological flocculant according to claim 3, which is characterized in that
The constituent of the seed liquid culture medium is as follows：Glucose 5g/L, yeast powder 20g/L, epsom salt 0.5g/L, phosphoric acid hydrogen
Dipotassium 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, the condition of culture of seed liquor are 170-190rpm, and 29-31 DEG C overnight
Culture.
5. application of the steady bacillus in conversion sweet potato stalk production biological flocculant according to claim 3, which is characterized in that
It is described fermentation liquid culture medium constituent be：Sweet potato stalk hydrolysate 300mL/L, yeast powder 20g/L, epsom salt
0.5g/L, dipotassium hydrogen phosphate 2g/L, potassium dihydrogen phosphate 5g/L, sodium chloride 2.4g/L, fermentation condition 170-190rpm, 33-35
℃。
6. application of the steady bacillus in conversion sweet potato stalk production biological flocculant according to claim 5, which is characterized in that
The preparation process of the sweet potato stalk hydrolysate is：Sweet potato stalk and 1.7% sulfuric acid solution are pressed into solid-to-liquid ratio 1:10 ratio is mixed
After conjunction, sour water solution 2h in 121 DEG C of high-pressure sterilizing pots；After cooling, by mixture after 10000rpm is centrifuged 10min, supernatant is collected
Liquid is added Ca (OH)2PH to 7.0 is adjusted, then supernatant is collected through 10000rpm centrifugation 10min again, obtains sweet potato stalk water
Solve object.
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| CN201810817291.1A CN108841765B (en) | 2018-07-24 | 2018-07-24 | Sterobacter and application thereof in production of bioflocculant by transforming sweet potato straws |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205268A (en) * | 2019-06-04 | 2019-09-06 | 江苏师范大学 | One plant of microbacterium and its conversion reed straw hydrolysate prepare the application in microbial flocculant |
| CN111808774A (en) * | 2020-07-24 | 2020-10-23 | 江苏师范大学 | Recyclable oxygen indicator and preparation method and application thereof |
| CN116656528A (en) * | 2023-03-15 | 2023-08-29 | 天津科技大学 | Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238843A (en) * | 2015-11-09 | 2016-01-13 | 成都信息工程大学 | Microbial flocculant, preparation method and application thereof |
-
2018
- 2018-07-24 CN CN201810817291.1A patent/CN108841765B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238843A (en) * | 2015-11-09 | 2016-01-13 | 成都信息工程大学 | Microbial flocculant, preparation method and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205268A (en) * | 2019-06-04 | 2019-09-06 | 江苏师范大学 | One plant of microbacterium and its conversion reed straw hydrolysate prepare the application in microbial flocculant |
| CN111808774A (en) * | 2020-07-24 | 2020-10-23 | 江苏师范大学 | Recyclable oxygen indicator and preparation method and application thereof |
| CN116656528A (en) * | 2023-03-15 | 2023-08-29 | 天津科技大学 | Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same |
| CN116656528B (en) * | 2023-03-15 | 2024-06-04 | 天津科技大学 | Pseudomonas and method for preparing deodorizing nitrogen-fixing bacteria agent by utilizing same |
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| CN108841765B (en) | 2021-04-06 |
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