CN104371951A - Serratia sp. A5 and uses thereof - Google Patents
Serratia sp. A5 and uses thereof Download PDFInfo
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- CN104371951A CN104371951A CN201410467628.2A CN201410467628A CN104371951A CN 104371951 A CN104371951 A CN 104371951A CN 201410467628 A CN201410467628 A CN 201410467628A CN 104371951 A CN104371951 A CN 104371951A
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- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/425—Serratia
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/023—Methane
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The present invention relates to Serratia sp. A5 and applications thereof. The Serratia sp. A5 is preserved in the China General Microbiological Culture Collection Center on August 28, 2014, and the preservation number is CGMCC No.9621. According to the present invention, the Serratia sp. A5 is isolated from soil, the suitable growth temperature is 25-35 DEG C, the pH value is 7.0-7.5, and the Serratia sp. A5 has the lipase production ability; and the Serratia sp. A5 can be applied for degrading grease, relieving the inhibition effect of the grease in food waste anaerobic fermentation, increasing the biogas yield by 15-30%, and effectively increasing anaerobic fermentation hydrolysis and biogas production efficiency of other waste containing fat and the like or waste water, and further provides important significance for promotion of harmless, resource and energy utilization of organic waste.
Description
Technical field
The present invention relates to a kind of Serratia A5 and uses thereof, the particularly application of Serratia A5 in changing food waste degraded, 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, and the characteristics such as with organic substances such as starch based, animal tallow classes for main component, these changing food waste amounts of having are large, high-moisture percentage, high organic content, harmless treatment rate is less than 5%.The water ratio of changing food waste, up to more than 80%, is not suitable for being directly used in several processing modes such as burning, landfill, compost and fodder.
Country requires recycling treatment to changing food waste process general thought, develops a circular economy, realizes recycle.And anaerobic fermentation technology to be organic waste innoxious, the effective way of resource utilization and recovery energy, be realize one of large effective means of dissolving of changing food waste.Changing food waste contains the nutritive substances such as abundant fat, starch, Mierocrystalline cellulose, protein and inorganic salt, and be the good raw material of anaerobically fermenting, its anaerobically fermenting process has become study hotspot in recent years.
Containing a large amount of greases in changing food waste, these greases are the processes such as kitchen waste is decocted, boils, fries, exploded, wash dining set and produce, its complicated component, form of diverse, too high fat content can reduce anaerobic digestion of kitchen wastes methane phase efficiency, suppress anaerobic digestion process, for running reactor continuously, the residence time can be caused to increase, degradation problem under processing power.Therefore, by adding efficient functional microorganism before carrying out anaerobic fermentation of kitchen waste, oils degradation is become the available small-molecule substance of methane phase fermentative, then carry out anaerobically fermenting, can effectively improve raw material hydrolysis efficiency and system run all right.
Serratia (
serratiasp.) very wide in distributed in nature, be often occupy flora in water and soil, nearly all bacterial strain can at 10 ~ 36 DEG C, pH 5 ~ 9.Having now utilizes this characteristic of strain secretes lipase of Serratia to carry out the patent of industrial production or degraded, if patent CN200910047517.5 is by the separation of immobilized Serratia lipase catalysis methoxyphenyl glycidic acid methyl esters, the fermentation broth contents of its catalyzed reaction is simpler, the kind of catalytic substrate is single, make a big difference with the process of changing food waste or waste water, and the optimal pH of the Serratia lipase of this catalyzed reaction is that alkalescence (pH8.0 ~ 8.5) generally also has very big-difference in slightly acidic with changing food waste or waste water.Patent CN01813506.4 then gives the another kind of microorganism combining bacteria having the wastewater treatment of fatty substance for load, what this combination bacterium comprised main utilization is the degradation capability of acid-producing Klebsiella bacterium to fat, addition of a small amount of smell Serratia and Aeromonas hydrophila.The present invention removes mainly for fat content high in changing food waste, adopts a kind of Serratia A5 of high yield lipase just can reach pretreating effect, improves anaerobic fermentation of kitchen waste hydrolysis efficiency.
Summary of the invention
The object of the present invention is to provide novel a large amount of secretion lipase, under weak acid to neutrallty condition, the Serratia A5 of anaerobic degradation fat, this Serratia is specially adapted 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:
Serratia A5 of the present invention has carried out preservation on August 28th, 2014 at China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, and preserving number is: CGMCC No.9621.
The amount that Serratia A5 of the present invention secretes lipase is at least 35 U/mL.
The application of Serratia A5 of the present invention in preparation biological degradation microbial inoculum.
Biological degradation microbial inoculum of the present invention is used for changing food waste degraded oil removing or oil wastewater anaerobically fermenting.
Microbial inoculum of the present invention is the bacterium liquid containing Serratia A5, and described microbial inoculum is not less than 10 containing the amount of Serratia A5
9individual/mL, the inoculum size of described microbial inoculum is 5 ~ 20% of changing food waste fermented liquid or wastewater volume.
Serratia A5 of the present invention, is separated from soil, and LB substratum grows, and bacterium colony is circular, translucent, moistening, smooth surface, colony edge are neat.This bacterial strain is Gram-negative G-, and thalline is rod-short, single, paired or short catenation, thalline size (0.4 ~ 0.8) μm × (1.3 ~ 3.0) μm, and motion, has pod membrane, do not form gemma.Amphimicrobian, chemoheterotrophy, oxidase negative, M.R tests the positive, and VP tests the positive, hydrolyzable gelatin, utilizes propanedioic acid, and nitrate reduction is positive, utilizes glucose to produce sour aerogenesis, does not produce yellow pigment and haematochrome.Serratia A5 suitable growth temperature of the present invention is 25 ~ 35 DEG C, and pH value is 7.0 ~ 7.5.
Beneficial effect of the present invention is:
Serratia A5 of the present invention, under normal temperature and medium temperature condition, can produce lipase, and longer chain fatty acid is degraded to the small-molecule substances such as water-soluble glycerine, monoglyceride or diester.Secondly, Serratia A5 of the present invention, can be degraded to small molecules product, significantly improve the oil removal rate containing grease rubbish by the lipid in changing food waste.Moreover Serratia A5 of the present invention, except for anaerobic fermentation of kitchen waste, effectively can improve its changing food waste or the hydrolysis of oil-containing waste water anaerobically fermenting and produce biogas efficiency.Realization of the present invention, not only efficient solution can remove high fat content to the restraining effect of anaerobic fermentation of kitchen waste, improve changing food waste hydrolysis efficiency and production rate of methane, and to promotion containing innoxious, the resource utilization of fatty wastes and recovery energy significant, have practical economical, societal benefits and wide application prospect.
biological sample preservation information
Serratia A5, Classification And Nomenclature is
serratiasp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 28th, 2014, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, culture presevation number is CGMCC No. 9621.
Accompanying drawing explanation
fig. 1 is the phylogenetic tree of Serratia A5 of the present invention.
Embodiment
embodiment 1 Serratia A5 is separated and preservation
Serratia A5 is from soil, is separated obtains by gradient dilution method.
Concrete grammar is: the place of polluting from dining room, Hebei Academy of Sciences periphery leftovers, take 10 g(soil) earth, add in the triangular flask that 90 mL sterilized waters, band granulated glass sphere are housed, shaking table vibrates 30 min (150 r/min), stepwise dilution, makes 10
-5, 10
-6dilution suspension, respectively draws 0.1 mL wherein, is added on lipase primary dcreening operation substratum (adding tween-80) flat board, and coating is even, each process repetition 3 times.By above-mentioned culture dish, be placed in that 25 DEG C of constant temperature culture haloings are large and single bacterium colony of looking fast is transferred on LB test tube slant, in 4 DEG C of preservations.
Multiple sieve is carried out by enzyme activity determination method.Receiving in LB liquid nutrient medium by being separated the bacterial strain obtained, cultivating 48 ~ 72 h in 25 DEG C of incubators, fermented liquid is temperature 4 DEG C, and centrifugal 10 min under rotating speed 8000 r/min, get supernatant liquor and measure lipase activity.Measuring method adopts GB GB/T 23535-2009(lipase measurement method).The lipase activity obtaining strains A 5 reaches 30 ~ 45 U/mL.
Adopt substratum:
Lipase primary dcreening operation substratum: extractum carnis 5 g, peptone 10 g, NaCl 5 g, tween 80 2 g, sweet oil 10 mL, agar powder 10 g, 1000 mL deionized water constant volumes, pH7.0 ~ 7.5.
LB substratum: yeast extract 5.0 g, Tryptones 10.0 g, sodium-chlor 10.0 g, agar 15.0 g, water 1000 mL, pH7.0 ~ 7.4.
This strains A 5, LB substratum grows, and bacterium colony is circular, surrounding is smooth, moistening, white, not translucent, colony edge is neat.Thalline rod-short, motion, thalline size (0.4 ~ 0.8) μm × (1.3 ~ 3.0) μm, Gram-negative, do not produce gemma, amphimicrobian, chemoheterotrophy, oxidase negative, M.R tests the positive, VP tests the positive, hydrolyzable gelatin, utilizes propanedioic acid, and nitrate reduction is positive, utilize glucose to produce sour aerogenesis, do not produce yellow pigment and haematochrome.
Bacterial strain 16S rDNA sequential analysis: choose the bacterium liquid being in logarithmic phase, extracts strain gene group DNA by Tian Gen company bacterial genomes DNA extraction kit, with it for template, carries out 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 of 3 min, 30 circulations extend; 72 DEG C of 10 min.
By Beijing, Tian Gen company carries out PCR primer order-checking, and its 16S rDNA sequence is shown in sequence table seq1.With DNAMAN software building phylogenetic tree (specifically seeing Fig. 1) after having checked order.According to strain morphology, Physiology and biochemistry and 16S rDNA sequential analysis, strains A 5 be accredited as Serratia (
serratiasp.).
embodiment 2 Serratia A5 enzyme activity determination
By be separated the Serratia A5 that obtains with can produce lipase serratia marcescens (
serratia marcescens), liquefied Serratia (
serratia liqufaciens), smell Serratia (
serratia odorifera), serratia rubida (
serratia rubidaea) receive in LB liquid nutrient medium respectively, cultivate 48 h in 30 DEG C of shaking table shaking culture, fermented liquid is temperature 4 DEG C, and centrifugal 10 min under rotating speed 8000 r/min, get supernatant liquor and measure lipase activity.
Measuring method adopts respectively: GB GB/T 23535-2009.
Enzyme is lived and is defined: 1 g1 mL liquid enzymes (or solid enzyme powder), under certain temperature and pH condition (the present embodiment adopts 40 DEG C, pH 7.0), 1 min hydrolysis substrate (this patent take sweet oil as substrate) produces the titratable lipid acid of 1 umol, be 1 enzyme activity unit, with " U/mL(U/g) " represent.
As shown in Table 1, Serratia A5 yielding lipase vigor is 35-45 U/mL, serratia marcescens, liquefied Serratia and smell Serratia yielding lipase vigor difference are not remarkable, and at 10-23 U/mL, serratia rubida yielding lipase vigor is relatively low is significantly higher than other a few strain Serratias.Illustrate that the Serratia A5 yielding lipase vigor that this patent filters out is significantly higher than other several Serratia strains.
the different Serratia yielding lipase of table 1 is lived and is compared
the application of embodiment 3 ~ 7 Serratia A5 in anaerobic fermentation of kitchen waste
First loaded in triangular flask by changing food waste, if 6 process, 5 experimental group, by 5% ~ 20% access Serratia A5 bacterium liquid, bacterium liquid is containing total plate count>=10
9individual/mL; 1 blank group, does not add Serratia A5.Shaking table shaking culture 13 ~ 24 h, the oily matter completely dissolve in experimental group, and in control group, oily matter has no minimizing.
Adopt 5 L full-mixing type anaerobic fermentation units again, below respectively organizing changing food waste is raw material, inoculum derives from normal fermentation methane-generating pit, inoculum size 25 ~ 30%(volume ratio), final anaerobic fermented liquid TS is that 5 ~ 8%(TS is also known as dry substance concentration, refer to a certain amount of fermented feed liquid to be placed in 105 DEG C of baking ovens, dry to constant weight, dry the per-cent that material accounts for gross weight).Middle temperature (35 ± 1 DEG C) condition bottom fermentation, fermentation period 30d, with the changing food waste fermentation without Serratia A5 process for contrast, found that and can shorten fermentation starting time 2-3 days through Serratia process, improve biogas output 15 ~ 30%, specifically in table 2.
the Application Example 3-7 of table 2 Serratia A5 in anaerobic fermentation of kitchen waste
SEQUENCE LISTING
<110> Biology Inst., Hebei Academy of Sciences
<120> Serratia A5 and uses thereof
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1442
<212> DNA
<213> Serratia 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 Serratia A5, this Serratia has carried out preservation on August 28th, 2014 at China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, and preserving number is: CGMCC No.9621.
2. a kind of Serratia A5 according to claim 1, is characterized in that the amount of its secretion lipase is at least 35 U/mL.
3. the application of a kind of Serratia A5 according to claim 1 in preparation biological degradation microbial inoculum.
4. the application of a kind of Serratia A5 according to claim 3 in preparation biological degradation microbial inoculum, is characterized in that described biological degradation microbial inoculum is for changing food waste degraded oil removing or oil wastewater anaerobically fermenting.
5. the application of a kind of Serratia A5 according to claim 4 in preparation biological degradation microbial inoculum, it is characterized in that described microbial inoculum is the bacterium liquid containing Serratia A5, described microbial inoculum is not less than 10 containing the amount of Serratia A5
9individual/mL, the inoculum size of described microbial inoculum is 5 ~ 20% of changing food waste fermented liquid or wastewater volume.
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Cited By (6)
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|---|---|---|---|---|
| CN104845905A (en) * | 2015-03-26 | 2015-08-19 | 北京北华中清环境工程技术有限公司 | High-efficiency composite inocula for removing salad oil-containing waste water COD and application thereof |
| CN104862244A (en) * | 2015-03-26 | 2015-08-26 | 北京北华中清环境工程技术有限公司 | Efficient composite bacterial agent for removing mixed grease-containing wastewater COD, and applications thereof |
| CN110283758A (en) * | 2019-07-25 | 2019-09-27 | 常州大学 | One plant of grease degrading strain IUMR B55 and its application |
| CN111826326A (en) * | 2020-08-05 | 2020-10-27 | 江西农业大学 | Bacterial strain for degrading lignin in papermaking wastewater and screening method and application thereof |
| CN111979160A (en) * | 2020-09-17 | 2020-11-24 | 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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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845905A (en) * | 2015-03-26 | 2015-08-19 | 北京北华中清环境工程技术有限公司 | High-efficiency composite inocula for removing salad oil-containing waste water COD and application thereof |
| CN104862244A (en) * | 2015-03-26 | 2015-08-26 | 北京北华中清环境工程技术有限公司 | Efficient composite bacterial agent for removing mixed grease-containing wastewater COD, and applications thereof |
| 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 |
| CN110283758A (en) * | 2019-07-25 | 2019-09-27 | 常州大学 | One plant of grease degrading strain IUMR B55 and its application |
| CN110283758B (en) * | 2019-07-25 | 2022-06-10 | 常州大学 | Grease degrading bacterium IUMR B55 and application thereof |
| CN111826326A (en) * | 2020-08-05 | 2020-10-27 | 江西农业大学 | Bacterial strain for degrading lignin in papermaking wastewater and screening method 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 |
| CN111979160A (en) * | 2020-09-17 | 2020-11-24 | 西南大学 | Serratia marcescens strain KW-P1 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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