CN104371951B - A kind of Serratieae A5 and application thereof - Google Patents
A kind of Serratieae A5 and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of Serratieae（Serratiasp.）A5 and its application.This Serratieae has carried out preservation in China Committee for Culture Collection of Microorganisms's General Microbiological Culture collection on 28th in August in 2014, and preserving number is：CGMCC No. 9621.The Serratieae A5 that the present invention provides is isolatable from soil, 25～35 DEG C of suitable growth temperature, and pH value is 7.0～7.5, has yielding lipase ability.Serratieae A5 in the present invention is applied to oils degradation, releases the inhibitory action of oils and fatss in anaerobic fermentation of kitchen waste, improves methane gas producing amount 15～30%.Garbage or anaerobic waste water hydrolysis fermentation and the product biogas efficiency of other fatty grades also can be effectively improved, resource innoxious to promotion organic waste and recovery energy are significant.
Description
Technical field
The present invention relates to a kind of Serratieae A5 and application thereof, particularly to Serratieae A5 in changing food waste degraded
Application, belongs to biological technical field.
Background technology
According to statistics, Chinese city produces changing food waste every year more than 6,000 ten thousand tons, organic with starch based, Animal fat class etc.
Material is main component, and these changing food waste amounts of having are big, the characteristic such as high-moisture percentage, high content of organics, harmless treatment rate
Less than 5%.The moisture content of changing food waste up to more than 80%, is not suitable for being directly used in burning, landfill, compost and fodder etc. several
Processing mode.
Country processes general thought to changing food waste and requires recycling treatment, develops a circular economy, realizes recycling.And
Anaerobic fermentation technology is that organic waste is innoxious, the effective way of resource and recovery energy, is that to realize changing food waste big
One of effective means that ancestor dissolves.Changing food waste contains the nutrition such as abundant fat, starch, cellulose, protein and inorganic salt
Material, is the good raw material of anaerobic fermentation, and its anaerobic fermentation processes the study hotspot having become in recent years.
Substantial amounts of oils and fatss are contained, these oils and fatss are that kitchen waste is decocted, boils, frying, exploding, washing tableware and waited in changing food waste
Journey and produce, its complicated component, form of diverse, too high fat content can reduce anaerobic digestion of kitchen wastes methane phase effect
Rate, suppresses anaerobic digestion process, runs reactor for continuous, and the time of staying can be caused to increase, the problems such as disposal ability declines.
Therefore, by adding efficient functional microorganism before carrying out anaerobic fermentation of kitchen waste, oils degradation is become methane phase fermentation
The available small-molecule substance of microorganism species, then carry out anaerobic fermentation, raw material hydrolysis efficiency and system operation can be effectively improved
Stability.
Serratia（Serratiasp.）Very wide in distributed in nature, it is often to occupy flora, Ji Husuo in water and soil
Some bacterial strains can be in 10～36 DEG C, pH 5～9.Carried out using this characteristic of strain secretes Digestive Enzyme of Serratieae now
Commercial production or the patent of degraded, such as patent CN200910047517.5 are catalyzed methoxy benzene by immobilized Serratia lipase
The separation of base glycidic acid methyl ester, the fermentation broth contents of its catalytic reaction are simpler, the single varieties of catalytic substrate,
Process with changing food waste or waste water makes a big difference, and the optimum pH of the Serratia lipase of this catalytic reaction is alkali
Property（pH8.0~8.5）Generally also there is very big difference with changing food waste or waste water in faintly acid.Patent CN01813506.4 is then given
The microorganism combining bacteria of another kind of wastewater treatment for being loaded with fatty material, this combination strain include mainly using be to produce
Sour klebsiella, to fatty degradation capability, addition of a small amount of abnormal smells from the patient Serratieae and Aeromonas hydrophila.The present invention is main
Remove for fat content high in changing food waste, pretreatment effect just can be reached using a kind of Serratieae A5 of high yield Digestive Enzyme
Really, improve anaerobic fermentation of kitchen waste hydrolysis efficiency.
Content of the invention
It is an object of the invention to provide new a large amount of secretion Digestive Enzyme, under weak acid to neutrallty condition, anaerobic degradation
The Serratieae A5 of fat, this Serratieae is particularly well-suited to the degradation treatment of fat in food garbage or waste water.
For achieving the above object, the technical solution used in the present invention is：
The Serratieae A5 of the present invention is commonly micro- in China Committee for Culture Collection of Microorganisms in August in 2014 28 days
Biological inoculum collection has carried out preservation, and preserving number is：CGMCC No.9621.
The amount that the Serratieae A5 of the present invention secretes Digestive Enzyme is at least 35 U/mL.
Application in preparation biodegradation microbial inoculum for the Serratieae A5 of the present invention.
Biodegradation microbial inoculum of the present invention is used for changing food waste degraded oil removing or oil wastewater anaerobic fermentation.
Microbial inoculum of the present invention is the bacterium solution containing Serratieae A5, and the amount that described microbial inoculum contains Serratieae A5 is not less than
109Individual/mL, the inoculum concentration of described microbial inoculum is the 5～20% of changing food waste fermentation liquid or wastewater volume.
Serratieae A5 of the present invention, is isolatable from soil, grows in LB culture medium, and bacterium colony is circular, translucent, wet
Profit, smooth surface, colony edge are neat.This bacterial strain is Gram-negative G-, and thalline is in rod-short, single, paired or short
Catenation, thalline size（0.4～0.8）μm×（1.3～3.0）μm, motion, there is pod membrane, do not form spore.Amphimicrobian,
Chemoheterotrophy, oxidase negative, M.R test is positive, and VP test is positive, hydrolyzable gelatin, utilizes malonic acid, nitrate reduction
The positive, produces sour aerogenesis using glucose, does not produce flavochrome and red pigment.Serratieae A5 suitable growth temperature of the present invention
For 25～35 DEG C, pH value is 7.0～7.5.
Beneficial effects of the present invention are：
The Serratieae A5 of the present invention, under room temperature and medium temperature condition, can produce Digestive Enzyme, long-chain fatty acid is degraded to
The small-molecule substances such as water-soluble glycerol, monoglyceride or diester.Secondly, the Serratieae A5 of the present invention, can be by kitchen rubbish
Oils in rubbish are degraded to small molecule product, significantly improve the oil removal rate containing oils and fatss rubbish.Furthermore, the Serratieae of the present invention
A5 removes for anaerobic fermentation of kitchen waste, can effectively improve its changing food waste or the hydrolysis of oil-containing waste water anaerobic fermentation and produce biogas
Efficiency.The realization of the present invention, not only can effectively release the inhibitory action to anaerobic fermentation of kitchen waste for the high fat content, improve
Changing food waste hydrolysis efficiency and production rate of methane, and to promoting containing fatty wastes are innoxious, resource and recovery energy
Significant, there are practical economical, societal benefits and wide application prospect.
Biological sample preservation information
Serratieae A5, Classification And Nomenclature isSerratiaSp., it is preserved in China Microbiological bacterium within 28th in August in 2014
Plant preservation administration committee common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of academy of science of state, postcode 100101, culture presevation number is CGMCC No. 9621.
Brief description
Fig. 1 is the phylogenetic tree of Serratieae A5 of the present invention.
Specific embodiment
Embodiment 1 Serratieae A5 separates and preservation
Serratieae A5 is from soil, is separated by gradient dilution method and obtains.
Concrete grammar is：From the place of Hebei Academy of Sciences dining room periphery leftovers pollution, weigh 10 g（Soil）Earth, plus
Enter equipped with 90 mL sterilized water, the triangular flask with bead, shaking table vibrates 30 min (150 r/min), stepwise dilution, system
Become 10-5、10-6Dilution suspension, respectively draws 0.1 mL wherein, is added to Digestive Enzyme primary dcreening operation culture medium（Plus tween 80）On flat board,
Uniformly, each processes and repeats 3 times for coating.By above-mentioned culture dish, it is placed in 25 DEG C of constant temperature culture haloings greatly and grows fast single bacterium
Fall to being transferred on LB test tube slant, preserve in 4 DEG C.
Secondary screening is carried out by enzyme activity determination method.It is connected to separating the bacterial strain obtaining in LB fluid medium, cultivate in 25 DEG C
48～72 h cultivated by case, and fermentation liquid, in 4 DEG C of temperature, is centrifuged 10 min under rotating speed 8000 r/min, take supernatant to measure fat
Enzyme activity.Assay method adopts GB GB/T 23,535 2009（Lipase measurement method）.Obtain the lipase activity of strains A 5
Power reaches 30～45 U/mL.
Using culture medium：
Digestive Enzyme primary dcreening operation culture medium：Carnis Bovis seu Bubali cream 5 g, peptone 10 g, NaCl 5 g, Tween 80 2 g, olive oil 10
ML, agar powder 10 g, 1000 mL deionized water constant volume, pH7.0～7.5.
LB culture medium：Yeast extract 5.0 g, tryptone 10.0 g, sodium chloride 10.0 g, agar 15.0 g, water
1000 mL, pH7.0～7.4.
This strains A 5, grows in LB culture medium, and bacterium colony is circular, surrounding is smooth, moistening, white, not translucent, bacterium colony side
Edge is neat.Thalline rod-short, motion, thalline size（0.4～0.8） μm×（1.3～3.0）μm, Gram-negative, do not produce
Spore, amphimicrobian, chemoheterotrophy, oxidase negative, M.R test is positive, and VP test is positive, hydrolyzable gelatin, utilizes third
Diacid, nitrate reduction is positive, produces sour aerogenesis using glucose, does not produce flavochrome and red pigment.
Bacterial strain 16S rDNA sequence analysis：Choose the bacterium solution being in exponential phase, with Tiangeng company bacterial genomes DNA
Extracts kit extracts strain gene group DNA, with it as template, enters performing PCR amplification.
Amplimer is bacterial universal primers：
Forward primer is 5 '-AGAGTTTGACC TGGCTCAG-3 ',
Reverse primer is Pr：5′-ACGGCTACCTTGTTACGACT-3′.
PCR response procedures：Carry out denaturation 4 min at 95 DEG C；95 DEG C of 1 min, 52 DEG C of 1 min, 72 DEG C 3
Min, 30 circulations extend；72 ℃ 10 min.
By Beijing, Tiangeng company carries out PCR primer sequencing, and its 16S rDNA sequence is shown in sequence table seq1.After sequencing completes
With DNAMAN software building phylogenetic tree（It is specifically shown in Fig. 1）.Divided according to strain morphology, Physiology and biochemistry and 16S rDNA sequence
Analysis, strains A 5 is accredited as Serratieae（Serratiasp.）.
Embodiment 2 Serratieae A5 enzyme activity determination
The Serratieae A5 obtaining and the serratia marcescens that Digestive Enzyme can be produced will be separated（Serratia marcescens）, liquefied Serratia（Serratia liqufaciens）, abnormal smells from the patient Serratieae（Serratia odorifera）, serratia rubida（Serratia rubidaea）It is coupled with LB fluid medium, in 30 DEG C of shaking tables
Shaken cultivation cultivates 48 h, and fermentation liquid, in 4 DEG C of temperature, is centrifuged 10 min under rotating speed 8000 r/min, takes supernatant to measure fat
Fat enzyme activity.
Assay method is respectively adopted：GB GB/T 23,535 2009.
Enzyme activity defines：1 g1 mL liquid enzymes（Or solid enzyme powder）, under the conditions of uniform temperature and pH（The present embodiment adopts
40 ℃、pH 7.0）, 1 min hydrolysis substrate（This patent is with olive oil as substrate）Produce the titratable fatty acid of 1 umol,
It is 1 enzyme activity unit, with " U/mL（U/g）" represent.
As shown in Table 1, Serratieae A5 yielding lipase vigor is 35-45 U/mL, serratia marcescens, the husky Lei Shi of liquefaction
Bacterium and abnormal smells from the patient Serratieae yielding lipase vigor difference is not notable, in 10-23 U/mL, serratia rubida yielding lipase vigor
Relatively low it is significantly higher than other several plants of Serratieaes.Illustrate that the Serratieae A5 yielding lipase vigor that this patent filters out is notable
Higher than other several Serratieae strains.
The different Serratieae yielding lipase work of table 1 is compared
Application in anaerobic fermentation of kitchen waste for embodiment 3 ~ 7 Serratieae A5
Changing food waste is initially charged with triangular flask, if 6 process, 5 experimental grouies, by 5%～20% access Serratieae A5
Bacterium solution, bacterium solution contains total number of bacteria >=109Individual/mL；1 blank control group, is added without Serratieae A5.Shaking table shaken cultivation 13
～24 h, the grease in experimental group is wholly absent, and in matched group, grease has no minimizing.
Adopt 5 L full-mixing type anaerobic installation for fermentings again, above each group changing food waste is raw material, inoculum derives from normal sending out
Fermentation methane tank, inoculum concentration 25～30%（Volume ratio）, final anaerobic fermented liquid TS is 5～8%（TS also known as dry substance concentration, refer to by
A certain amount of fermented feed liquid is placed in 105 DEG C of baking ovens, dries to constant weight, dries the percentage ratio that material accounts for gross weight）.Middle temperature（35
±1 ℃）Condition bottom fermentation, fermentation period 30d, to ferment for comparison without the changing food waste that Serratieae A5 is processed, result is sent out
Now process through Serratieae and can shorten 2-3 days fermentation starting time, improve biogas output 15～30%, be specifically shown in Table 2.
Application Example 3-7 in anaerobic fermentation of kitchen waste for the table 2 Serratieae A5
SEQUENCE LISTING
<110>Biology Inst., Hebei Academy of Sciences
<120>A kind of Serratieae A5 and application thereof
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1442
<212> DNA
<213>Serratieae A5 (Serratia sp.)
<400> 1
atgaatccaa gtggtagcgc cctcccgaag gttaagctac ctacttcttt tgcaacccac 60
tcccatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg tagcattctg 120
atctacgatt actagcgatt ccgacttcac ggagtcgagt tgcagactcc gatccggact 180
acgacgtact ttatgaggtc cgcttgctct cgcgagttcg cttctctttg tatacgccat 240
tgtagcacgt gtgtagccct actcgtaagg gccatgatga cttgacgtca tccccacctt 300
cctccggttt atcaccggca gtctcctttg agttcccgcc attacgcgct ggcaacaaag 360
gataagggtt gcgctcgttg cgggacttaa cccaacattt cacaacacga gctgacgaca 420
gccatgcagc acctgtctca gagttcccga aggcactaag ctatctctag cgaattctct 480
ggatgtcaag agtaggtaag gttcttcgcg ttgcatcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcatttgag ttttaacctt gcggccgtac tccccaggcg 600
gtcgacttaa cgcgttagct ccggaagcca cgcctcaagg gcacaacctc caagtcgaca 660
tcgtttacag cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcacc 720
tgagcgtcag tctttgtcca gggggccgcc ttcgccaccg gtattcctcc agatctctac 780
gcatttcacc gctacacctg gaattctacc cccctctaca agactctagc ttgccagttt 840
caaatgcagt tcccacgtta agcgcgggga tttcacatct gacttaacaa accgcctgcg 900
tgcgctttac gcccagtaat tccgattaac gcttgcaccc tccgtattac cgcggctgct 960
ggcacggagt tagccggtgc ttcttctgcg agtaacgtca atgcacagtg ctattaacac 1020
tgaacccttc ctcctcgctg aaagtgcttt acaacccgaa ggccttcttc acacacgcgg 1080
catggctgca tcaggcttgc gcccattgtg caatattccc cactgctgcc tcccgtagga 1140
gtctggaccg tgtctcagtt ccagtgtggc tggtcatcct ctcagaccag ctagggatcg 1200
tcgcctaggt gagccattac cccacctact agctaatccc atctgggcac atctgatggc 1260
atgaggcccg aaggtccccc actttggtcc gaagacgtta tgcggtatta gctaccgttt 1320
ccagtagtta tccccctcca tcaggcagtt tcccagacat tactcacccg tccgccgctc 1380
gtcacccgga gagcaagctc tcctgtgcta ccgctcgact gcatgtgtag ctgccgccat 1440
gc 1442
Claims (5)
1. a kind of Serratieae（Serratia sp.）A5, this Serratieae is in August in 2014 28 days in Chinese microorganism strain
Preservation administration committee General Microbiological Culture collection has carried out preservation, and preserving number is：CGMCC No.9621.
2. a kind of Serratieae A5 according to claim 1 is it is characterised in that the amount of its secretion Digestive Enzyme is at least 35 U/
mL.
3. application in preparation biodegradation microbial inoculum for a kind of Serratieae A5 described in claim 1.
4. a kind of Serratieae A5 according to claim 3 preparation biodegradation microbial inoculum in application it is characterised in that
Described biodegradation microbial inoculum is used for changing food waste degraded oil removing or oil wastewater anaerobic fermentation.
5. a kind of Serratieae A5 according to claim 4 preparation biodegradation microbial inoculum in application it is characterised in that
Described microbial inoculum is the bacterium solution containing Serratieae A5, and the amount that described microbial inoculum contains Serratieae A5 is not less than 109Individual/mL, described bacterium
The inoculum concentration of agent is the 5～20% of changing food waste fermentation liquid or oil wastewater volume.
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| CN104862244B (en) * | 2015-03-26 | 2017-12-19 | 北京北华中清环境工程技术有限公司 | A kind of high efficiency composition microbial inoculum for removing the oil wastewater COD containing mixing and its application |
| CN104845905B (en) * | 2015-03-26 | 2018-02-16 | 北京北华中清环境工程技术有限公司 | The high efficiency composition microbial inoculum of removal COD containing salad oil wastewater a kind of and its application |
| CN110283758B (en) * | 2019-07-25 | 2022-06-10 | 常州大学 | Grease degrading bacterium IUMR B55 and application thereof |
| CN111826326B (en) * | 2020-08-05 | 2023-01-03 | 江西农业大学 | Bacterial strain for degrading lignin in papermaking wastewater and screening method and application thereof |
| CN111979160B (en) * | 2020-09-17 | 2022-11-01 | 西南大学 | Serratia marcescens strain KW-P1 and application thereof |
| CN116355782A (en) * | 2022-07-15 | 2023-06-30 | 海南大学 | Serratia rubra and application thereof |
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| CN101503683A (en) * | 2009-03-13 | 2009-08-12 | 华东理工大学 | Magnetic nano particle immobilized Serratieae lipase, preparation and use |
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