CN106222107B - One plant of extreme thermophilic bacterium from pig farm waste - Google Patents
One plant of extreme thermophilic bacterium from pig farm waste Download PDFInfo
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- CN106222107B CN106222107B CN201610633494.6A CN201610633494A CN106222107B CN 106222107 B CN106222107 B CN 106222107B CN 201610633494 A CN201610633494 A CN 201610633494A CN 106222107 B CN106222107 B CN 106222107B
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12R2001/07—Bacillus
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
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Abstract
The invention discloses one plant of extreme thermophilic bacteriums from pig farm waste.The bacterium is obtained by enrichment culture, primary dcreening operation, secondary screening etc., through Gram's staining it is found that being gram-positive short, thallus is single or arranges in pairs, brood cell's subcircular, and proximal end is raw, and sporangiocyst expands.Pass through the performance detection of this bacterial strain, display, cultivation field waste of this bacterial strain energy efficient degradation containing dead livestock and poultry is organic fertilizer, i.e. smokeless, odorless, without sewage discharge, it is able to achieve recycling for organic waste again, to reduce livestock and poultry cultivation pollution on the environment, it is one and meets technology that is environmentally protective, guaranteeing food safety.
Description
Technical field
The present invention relates to industrial microorganism fields, more specifically, it relates to one plant from the extremely thermophilic of pig farm waste
Hot bacterium and application.
Background technique
Thermophilic Bacteria (is commonly called as high temperature bacterium, also referred to as thermophilic microorganism), is the microorganism of a kind of life in the high temperature environment, extensively
It is general with being distributed in hot spring, compost, geothermal area soil, Volcanic Region and submarine volcano etc..In containing for Yellowstone National Park, USA
In sulphur hot spring, it was once separated to one plant of thermophilic facultative autotrophy bacterium --- acid heat sulfolobus solfataricus (Sulfolobus), they
Can higher than 90 DEG C at a temperature of grow.Thermophilic bacteria is not single Pseudomonas or flora, but be distributed widely in it is various not
With flora in, some of them thermophilic bacteria, with being all Thermophilic Bacteria in bacterium, such as red thermophilic bacteria (
Rhodothermus), Desulfotomaculum (Desu1. fotomaeulum), the curved bacterium of rose (Roseiflexus), thermophilic good
Oxygen bacillus (Thermoaerobium), thermophilic anaerobic bacillus (Thermoanaerobaeterium), thermophilic acidophilic beam hanger bacterium (
Thermoleophilum), green curved bacterium (Ctdorolfexus), thermophilic silk bacterium (Thermothrix), thermophilic pipe bacterium (
Thermosipho), coccobacillus (Sphaembaeter) etc., the Pseudomonas for also having high temperature bacterium and mesophile and depositing.Such as gemma bar
Bacterium, unusual coccus (Deincooccus), false Flavimonas (Pseudoxanthomonas) etc..Thermophilic Bacteria is a kind of life
Microorganism in a high temperauture environment living can be divided into Thermophilic Bacteria five different monoids: heat-resisting according to their temperature capability
Bacterium, facultative thermophile, obligate thermophilic bacteria, Thermophilic Bacterium and hyperthermophile.
Thermophilic Bacterium: maximum growth temperature is higher than 70 DEG C, and optimum temperature is higher than 65 DEG C, and minimum growth temperature is higher than 40
℃.Up to the present, found that a variety of thermophilic microorganisms can generate heat-staple zytase successively, mainly
Thermotoga belong to some kinds, wherein better heat stability be from Thermotoga sp. FiSS3-B. 1,
The enzyme of Thermotoganeapolitana, Thermotoga maritime and Thermotogathermarum separation, they
Stability at 80-105 DEG C.
The processing of the special dead livestock and poultry of livestock and poultry cultivation field waste is long-standing problem, not only increases pressure to environmental protection,
And restrict the development of animal husbandry.Therefore the microorganism of cultivation field waste of the screening energy efficient degradation containing dead livestock and poultry has pole
Big meaning.
Summary of the invention
The invention aims to provide one plant of extreme thermophilic bacterium from pig farm waste.
The bacterium be heat bite bacillus amyloliquefaciens (Bacillus thermoamylovorans) HL8103, in
On June 15th, 2016, deposit number was CGMCC No.12613, address in China General Microbiological culture presevation administrative center preservation
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Application of the bacterium in degradation livestock and poultry cultivation field waste.
Culture medium:
(1) LB culture solution (L): yeast extract 10g, peptone 10g, beef extract powder 5g, NaCl10g, pH7.2,
Add distilled water to be settled to 1L to be made.
(2) it isolates and purifies solid medium: 20g agar powder being added when in LB culture solution.
(3) liquid fermentation medium: yeast extract 10g, peptone 10g, beef extract powder 5g, NaCl10g, pH7.2,
Add distilled water to be settled to 1L to be made.
Separation screening:
(1) enrichment culture: from pig farm waste compared with sample 0.5g is taken from center, addition fills 50mL LB culture medium
In 250mL conical flask, 2mL culture for 24 hours, is inoculated into fresh LB and trained by enrichment culture under the conditions of 60 DEG C, 120r/min
Base is supported, is cultivated with identical condition.So repeat 3 times.
(2) primary dcreening operation: enrichment culture object is crossed culture on isolating and purifying solid medium, and 60 DEG C of cultures are for 24 hours.Picking is raw
Long apparent bacterium colony carries out secondary screening.
(3) secondary screening: separation 3 times that primary dcreening operation bacterium colony is crossed on isolating and purifying solid medium, to obtain pure culture
Object, and eugonic bacterial strain is selected in the growing state for isolating and purifying solid medium according to strain.
(4) in the conical flask that volume is 250mL, LB culture the measurement of the growth performance of extreme thermophilic bacterium: is added
Liquid 100mL, inoculation 1mL bacteria suspension (OD=1) carry out the culture of different pH and temperature under the conditions of 150r/min respectively,
Measure its optimal growth conditions.
Conclusion:
For the extreme thermophilic bacterium that separation screening of the present invention obtains in 70 DEG C of culture 20h of LB culture medium, bacterium colony is round, white,
Protrusion, surface is smooth, wet, neat in edge.
The extreme thermophilic bacterium that separation screening of the present invention obtains belongs to heat and bites bacillus amylobacter, Gram-positive, the bacterial strain
Morphological feature are as follows: thallus is rod-shaped, straight or bending, 1.6-6.0 μm of 0.3-0.5 μ m, single or arrangement in pairs, brood cell's subcircular,
Proximal end is raw, and sporangiocyst expands.Through detecting, which can be resistant to 90 DEG C of high temperature, and optimum growth temperature is 70 DEG C,
The most suitable growth pH value is 6.8-7.3.
The invention has the advantages that since the extreme thermophilic bacterium can be resistant to 90 DEG C of high temperature, thus can be easily
Oxygen concentration is controlled by controlling temperature, reduces production technology and equipment requirement；It can be in very simple Zymolysis Equipment (such as nothing
Antiforeign bacteria polluting device) in, it is grown using the cultivation field waste containing dead livestock and poultry as carbon nitrogen source；Reduce equipment investment and hair
Ferment substrate cost；Other non-high-temperature resistant bacteriums can also be killed by improving temperature, to guarantee target thallus unicity.
Detailed description of the invention
Fig. 1 is aspect graph under HL8103 bacterium colony natural light.
Fig. 2 is strain morphology figure under HL8103 bacterium Electronic Speculum.
Fig. 3 is the systematic growth that HL8103 is constructed based on 16S rDNA gene order with Neighbor-Joining method
Tree.
Specific embodiment
Below in conjunction with specific embodiment to the detailed description of the invention.
Embodiment 1
Process flow:
Acquisition sample → sample pretreatment → enrichment culture → selected high-temperature resistant strain → growth performance measurement → best
Grow measurement → optimum growth temp measurement → bacterium morphologic observation → acquisition HL8103 bacterial strain of pH value.
The separating screening method of extreme thermophilic bacterium of the present invention heats separation method using gradient and screens to obtain,
Method includes the following steps:
Acquisition sample: acquisition pig farm and the accumulation of pig slaughtering field waste are compared with taking product at center；
Sample pretreatment: it takes above-mentioned sample to be fitted into the container for having sterilized and being loaded with sterile water, is acutely shaked after sealing
10min stands suspension, and after taking supernatant to be centrifuged 3min, taking upper layer bacterium solution is bacteria suspension, spare；
Enrichment culture: it takes 10 μ L of bacteria suspension to be inoculated in 250mL LB culture solution respectively, is placed in 50 DEG C of insulating boxs and trains
Concussion 48h is supported, expansion bacteria culture fluid is obtained；
Step by step ladder heating screening: take 10 μ L expand bacteria culture fluid be inoculated in 250mL LB culture solution, by temperature by
Grade, which is risen progressively, is cultivated, i.e., latter temperature rank inoculation bacterium solution used is derived from the bacterium solution of previous temperature rank culture；
Selected high temperature resistant bacterium: under cultivation temperature, the LB culture solution after observation inoculation bacterium solution then determines if appearance is muddy
The bacterium can grow at this temperature, on the contrary then exclude；
Selected high-temperature resistant strain: resistance to 50 DEG C or more of bacterium solution is taken, with normal saline dilution (10-3), and it is spread evenly across LB
In culture medium, 70 DEG C of culture 20h；Picking has the single colonie of different shape feature, in expand respectively on 70 DEG C of LB culture mediums it is numerous after,
Add 20% glycerol to mix with complete nutrition fluid nutrient medium, is saved under the conditions of -80 DEG C；
The measurement of optimum growh pH value: the LB culture solution of different pH value is prepared, 50mL is respectively taken to be dispensed into volume 250mL
Conical flask in, be inoculated with 0.5mL bacteria suspension, 70 DEG C, cultivate 20h under the conditions of 150r/min, blank made with LB culture solution, in wave
The optical density of bacteria suspension is measured at long 540nm.
The measurement of optimum growth temp: the LB culture solution 50ml of pH7.2 is placed in 250mL conical flask, inoculation
0.5mL bacteria suspension cultivates 20h under the conditions of 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C and 90 DEG C respectively, in wave
Its OD value is measured at long 540nm.
The oscillation frequency of the culture concussion is 150r/min.
Embodiment 2: the identification method of extreme thermophilic bacterium of the invention, comprising the following steps:
(1) colonial morphology and strain morphology of extreme thermophilic bacterium are observed, extreme thermophilic bacterium of the present invention is in LB culture medium
70 DEG C of culture 20h, colony morphology characteristic are as follows: round, white, protrusion, surface is smooth, wet, neat in edge.(Fig. 1)
Extreme thermophilic bacterium of the present invention belongs to heat and bites bacillus amylobacter, Gram-positive, the strain morphology feature are as follows: bacterium
Body rod shape, straight or bending, 1.6-6.0 μm of 0.3-0.5 μ m, single or arrangement in pairs, brood cell's subcircular, proximal end is raw, and sporangiocyst is swollen
Greatly.(Fig. 2)
(2) bacterial strain physio-biochemical characteristics.
(3) sequence information and its phylogenetic analysis of DNA.
Using MEGA5.0 software, ortho position cascade synthesis shows that bacterial strain HL8103 and related kind of 16S rDNA sequential system are sent out
Tree is educated, 1000 similarities is carried out and computes repeatedly, tree node is developed in Fig. 3 and only shows that Bootstrap value is greater than 50% numerical value,
Subscript " T " intermediate scheme bacterial strain, the 16S rDNA sequence of bacterial strain of the present invention is as shown in SEQ ID NO.1.By analysis, of the invention
Extreme thermophilic bacterium and the homologous similarity of bacillus respective standard bacterial strain of reference, therefore can 98% or more therewith
Tentatively judge that bacterial strain HL8103 belongs to bacillus.Strains tested HL8103 and type strain Bacillus
The homology of thermoamylovorans CNCM I-1378 16S rDNA sequence reaches 100%, therefore, identifies that HL8103 is
Heat bites bacillus amyloliquefaciens (Bacillus thermoamylovorans).
(4) according to the above results, identify that the bacterial strain is that heat bites bacillus amyloliquefaciens.
Embodiment 3: the preparation method of extreme thermophilic bacterium bacterium powder of the invention
The following steps are included:
(1) actication of culture:
The extreme thermophilic bacterium activation that 4 DEG C of inclined-planes are saved, it is lined respectively on corresponding solid medium, 70 DEG C,
Cultivate 20h；
Strain source: extreme thermophilic bacterium, voluntarily screening obtains, and is preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, number is CGMCC No.12613；
(2) seed culture:
Picking activation bacterium colony, by extreme thermophilic microbionation in seed culture medium carry out seed culture, 70 DEG C,
150r/min shaking table culture 20h；
By mass concentration include following components in above-mentioned seed culture medium:
Yeast extract 10g/L, peptone 10g/L, beef extract powder 5g/L, NaCl 10g/L, PH7.2；
(3) tank fermented and cultured on:
By the extreme thermophilic bacterium seed liquor obtained in previous step according in 10% inoculum concentration access fermentor, fermentation is trained
Supporting base is, ventilating ratio 1:1.05 identical as above-mentioned seed culture medium, 70 DEG C, 180r/min, collects bacterium solution after cultivating 20h；
(4) it is spray-dried:
By bacterium solution using be spray-dried, using 5% skimmed milk power as protective agent it is dry powder, spray drying condition
Are as follows: 150 DEG C of intake air temperature, 90 DEG C of air outlet temperature, charging rate 15ml/min.
The bacterium solution is spray-dried to prevent the moisture absorption using sterile sealing bag packaging as after bacterium powder.
Embodiment 4: the application of extreme thermophilic bacterium bacterium powder of the invention
The following steps are included:
(1) dead livestock and poultry carries out cutting and grinding processing, according to " GB16548-1996 livestock and poultry disease meat corpse and products thereof is harmless
Change processing regulation " it is handled.
(2) farm's offal treatment containing dead livestock and poultry: by porcine tissue 40% of dying of illness after cutting and grinding, swine excrement 40%,
Husk 19%, extreme thermophilic bacterium bacterium powder 1% are uniformly mixed, are deposited in the hardened floor to keep off the rain with thickness 40cm, 0-6d is every
Turning is primary for 24 hours, and not turning later is again through 3-4d compost natural evaporation moisture, when moisture content is organic fertilizer up to standard within 30%.
A cycle is about 10 days.By this strain fermentation, energy cultivation field waste of the efficient degradation containing dead livestock and poultry is organic fertilizer, i.e.,
Smokelessly, odorless, without sewage discharge, and it is able to achieve recycling for organic waste, environment is caused to reduce livestock and poultry cultivation
Pollution, be one meet it is environmentally protective, guarantee food safety technology.It has broad application prospects.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>one plants of extreme thermophilic bacteriums from pig farm waste
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1422
<212> DNA
<213>sequence
<400> 1
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aacacgtggg taacctgcct gtaagaccgg gataactccg ggaaaccggt gctaataccg 120
gatagattat ctttccgcct ggagagataa ggaaagatgg cttttgccat cacttacaga 180
tgggcccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta 240
gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300
aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360
cgaagaaggt cttcggatcg taaagctctg ttgttaggga agaacaagta tcggaggaaa 420
tgccggtacc ttgacggtac ctgacgagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg atttattggg cgtaaagcgc gcgcaggcgg 540
tcctttaagt ctgatgtgaa atcttgcggc tcaaccgcaa gcggtcattg gaaactgggg 600
gacttgagtg cagaagagga aagcggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcggcttt ctggtctgta actgacgctg aggcgcgaaa 720
gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttggag ggtttccgcc cttcagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat ctcctgaccg 960
ccctggagac agggtcttcc cttcggggac aggatgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg gttctagttg 1080
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caaagggcag cgaagcggcg acgcatgagc gaatcccaga aaaccattct cagttcggat 1260
tgcaggctgc aactcgcctg catgaagccg gaatcgctag taatcgcgga tcagcatgcc 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac 1380
acccgaagtc ggtgaggtaa ccgcaaggag ccagccgccg aa 1422
Claims (2)
1. one plant of extreme thermophilic bacterium from pig farm waste, it is characterised in that: the bacterium is that heat bites starch gemma bar
Bacterium (Bacillus thermoamylovorans) HL8103, it is protected on June 15th, 2016 in China General Microbiological strain
Administrative center's preservation is hidden, deposit number is CGMCC No.12613.
2. application of the bacterium in degradation poultry waste as described in claim 1.
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| CN110964639A (en) * | 2019-12-09 | 2020-04-07 | 湖南金泥生物科技有限公司 | Strain screening method applied to pig raising fermentation bed and application |
| CN111808775B (en) * | 2020-07-24 | 2022-01-28 | 南京高新工大生物技术研究院有限公司 | High-temperature-resistant thermophilic bacillus amylovorus and application thereof |
| CN116200285B (en) * | 2021-12-01 | 2024-01-19 | 中国石油天然气股份有限公司 | Bacillus strain, microbial inoculum, metabolite, method and application thereof |
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| CN1662653A (en) * | 2002-05-14 | 2005-08-31 | 普拉克生化公司 | Method for the production of lactic acid or a salt thereof by simultaneous saccharification and fermentation of starch |
| CN103169153A (en) * | 2013-04-12 | 2013-06-26 | 上海烟草集团有限责任公司 | Method for improving internal quality of low-class tobacco through solid fermentation |
| CN103525714A (en) * | 2012-07-02 | 2014-01-22 | 中国科学院微生物研究所 | Method as well as special Bacillus thermoamylovorans for producing L-lactic acid |
| CN105601358A (en) * | 2015-10-22 | 2016-05-25 | 李德舜 | Microbial organic fertilizer for preventing and controlling strawberry red core disease, and production method thereof |
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| CN1662653A (en) * | 2002-05-14 | 2005-08-31 | 普拉克生化公司 | Method for the production of lactic acid or a salt thereof by simultaneous saccharification and fermentation of starch |
| CN103525714A (en) * | 2012-07-02 | 2014-01-22 | 中国科学院微生物研究所 | Method as well as special Bacillus thermoamylovorans for producing L-lactic acid |
| CN103169153A (en) * | 2013-04-12 | 2013-06-26 | 上海烟草集团有限责任公司 | Method for improving internal quality of low-class tobacco through solid fermentation |
| CN105601358A (en) * | 2015-10-22 | 2016-05-25 | 李德舜 | Microbial organic fertilizer for preventing and controlling strawberry red core disease, and production method thereof |
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| Cellulosic Fermentation Using Bacillus thermoamylovorans-Enabled Digested Sludge;Han, Chang-Lung Lay 等;《Journal of Environmental Engineering》;20051231(第12期);摘要 * |
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