CN107083343A - Bacterial strains of arthrobacterium HW 17 of one plant of degraded cellulose and application thereof - Google Patents
Bacterial strains of arthrobacterium HW 17 of one plant of degraded cellulose and application thereof Download PDFInfo
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- CN107083343A CN107083343A CN201710385917.1A CN201710385917A CN107083343A CN 107083343 A CN107083343 A CN 107083343A CN 201710385917 A CN201710385917 A CN 201710385917A CN 107083343 A CN107083343 A CN 107083343A
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- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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- C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
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Abstract
The present invention uses selective medium, method by orienting domestication, isolated plant height Cellulase Producing Bacteria is screened from the rotten wood of Enshitujianationalitymiaonationality autonomous region virgin forest, the bacterial strain is named as HW 17, belong to a kind of arthrobacterium, the bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University；Postcode 430072, preservation date is on March 30th, 2017, and deposit number is：CCTCC NO：M2017156；The 16S rDNA sequences of the bacterial strain are as shown in SEQ ID NO.1.The bacterial strains of HW 17 that the application is provided can be used for the industrial production organic fertilizer using chicken manure or pig manure, agricultural crop straw, vegetables rotten leaf etc. as raw material.In addition, the growth characteristics and cellulase-producing characteristic of the bacterial strains of HW 17 are perfectly clear, thus the bacterial strains of HW 17 can not only be used for the candidate strain of industrial production cellulase, it may also be used for the single cellulase of commercial production.
Description
Technical field
The present invention relates to a kind of arthrobacterium new strains, biodegradable fiber element, the more particularly to bacterial strain with agriculture and foster
It is the purposes in raw material production farm manure to grow the discarded object of industry, belongs to microbial technology field.
Background technology
Agricultural crop straw such as straw, straw and bagasse etc. are a kind of important renewable resources rich in cellulosic crops,
The annual stalk yield in the whole world be about 2,200,000,000 tons (Kim et al., 2004), China about 700,000,000 tons of agricultural crop straw of production (Xu every year
Outstanding person etc., 2008).Agricultural crop straw is in addition to fraction is for compost and as animal and fowl fodder, and most of arbitrarily to be burnt, this is not only
The resource of preciousness is wasted, and pollutes environment (Cheng et al., 2013).At present, microbial method degraded crops straw
Stalk is people's extensive concern (Gu Wenjie etc., 2012 with its most economical, maximally effective advantage；Zhang Jianfeng etc., 2016).In the past
Decades in, domestic and foreign scholars are found that the mucor in some microorganisms for being capable of degraded cellulose, such as Mycophyta successively
(Wen Zilan etc., 2015), whiterot fungi (Doddapaneni et al., 2005) etc., the streptomyces (Deng Hui in actinomycetes
Deng 2013；Feng Hongmei etc., 2016), Micromonospora (Kasusar et al., 2011) etc., the bacillus in bacterium class
(Wang Xianfeng etc., 2015), pseudomonas (still knowing beautiful jade etc., 2012) etc..In the practical application of microbial degradation cellulose, often
Frequently with the mode of cellulose degradation mixed bacterial, come the cellulose in agricultural crop straw of degrading with compost or in the form of making compost
(beam east is beautiful etc., 2009), the Period Process is longer and degrades insufficient.In the last few years, although induced mutations and genetic modification
The production that biotechnologies such as (Fujii et al., 2013) is gradually applied to during cellulose-degrading bacteria is transformed to improve bacterial strain is fine
The plain enzyme ability of dimension, but the bacterial strain obtained still has that yield of enzyme is few, the low problem of enzyme activity.Therefore, novel cellulose is screened
Degrading microorganism, finds optimal cellulose degradation approach, and the existing cellulose-degrading bacteria of genetic modification is particularly important.
The content of the invention
Object of the present invention is to provide the strong bacterium of a kind of High Cellulase Production, degradation property, the bacterial strain can be used for
Farm manure is produced by raw material of the discarded object of agricultural and aquaculture.
The present invention uses selective medium, the method by orienting domestication, and from Enshitujianationalitymiaonationality, autonomous region is original
Isolated plant height Cellulase Producing Bacteria is screened in the rotten wood of forest, the bacterial strain is named as HW-17, belongs to a kind of arthrobacterium
(Arthrobacter oryzae), the bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan
University；Postcode 430072, preservation date is on March 30th, 2017, and deposit number is：CCTCC NO：M2017156；The bacterial strain
16S rDNA sequences are as shown in SEQ ID NO.1.
Although HW-17 bacterial strains provided herein support as single cellulose-degrading bacteria to the degradation capability of cellulose
Not upper mixed culture is to the degradation capability of containing cellulose sample, but by the HW-17 inoculations to the fiber containing pig manure or chicken manure
In plain sample (v/w=1: 1), the stalk of containing cellulose has also obtained effective degraded, and has no other in the art and save
Report of the bacillus to cellulose degradation effect in the cellulose mixture sample containing chicken manure or pig manure.Thus the bacterium can be used for compost,
Available for the industrially production organic fertilizer using chicken manure or pig manure, agricultural crop straw, vegetables rotten leaf etc. as raw material.In addition, the HW-17
The growth characteristics and cellulase-producing characteristic of bacterial strain are perfectly clear, thus the HW-17 bacterial strains can not only be used for industrially producing fiber
The candidate strain of plain enzyme, it may also be used for the single cellulase of commercial production.
Embodiment
Detailed specific description is done to the present invention with reference to specific embodiment, but protection scope of the present invention not office
It is limited to following examples.
The screening and culture of bacterial strain
Using the rotten wood that is collected from the sisters mountain of Enshi State of Hubei Province seven as the material of bacterium in the application, use with carboxylic
Sodium carboxymethylcellulose pyce as single carbon source selective medium [sodium carboxymethylcellulose (CMC-Na) 10.0g/L,
KH2PO42.0g/L, NH4Cl5.0g/L, MgSO4·7H2O0.5g/L, pH 7.0-7.2], rotten wood is added into the selective medium
In, in taming 7d under 30 DEG C, 200r/min.Bacterium solution after domestication is diluted step by step, takes the bacterium solution of a small amount of different dilution factors uniform
It is coated on the Selective solid culture medium, in culture at 30 DEG C.The big bacterium colony of picking hydrolysis circle carries out line dilution 6 times, with
This is purified and strong single bacterium colony of cellulose degradation ability.
The bacterial strain physio-biochemical characteristics screened are as follows：
| Objects | Results | Objects | Results |
| Gram's staining | + | Alpha-lactose is utilized | - |
| VP is tested | - | Sucrose is utilized | + |
| Methyl red test | - | D- xylose utilizations | - |
| Starch Hydrolysis is tested | + | Maltose is utilized | + |
| Citrate test | - | Trehalose is utilized | + |
| Indole test | - | Rhamnose is utilized | + |
| Produce ammonia test | + | Sorbierite is utilized | - |
| H2S is tested | - | Mannitol is utilized | + |
| Hydrolysis of urea is tested | - | Methanol is utilized | + |
| H2O2Contact test | + | Ethanol is utilized | + |
| Gelatin liquefaction test | - | Oxalic acid is utilized | - |
| Malonate is tested | - | Glycerine is utilized | - |
| Glucose utilization | + | Sodium potassium tartrate tetrahydrate is utilized | - |
Note：+, positive hair should；-, negative reaction
The growth characteristics of the bacterial strain are as follows：
1. the preference of nitrogen source：In tryptone, peptone, yeast extract, casein, KNO3, NaNO2, NH4Cl and sweet
In this 8 kinds of nitrogen sources of propylhomoserin, utilizations of the Arthrobacter oryzae HW-17 to organic nitrogen source is best, particularly tryptone
2. the preference of carbon source：In glucose, sucrose, starch, trehalose, maltose, mannitol, ethanol, methanol, cellulose
In this 9 kinds of carbon sources, utilizations of the Arthrobacter oryzae HW-17 to ethanol is best
3. the most suitable growth pH：In the case where pH is 3,4,5,6,7,8,9 or 10, Arthrobacter oryzae HW-17 are in pH
To grow best under 7.
4. optimum growth temperature：Arthrobacter oryzae HW-17 can grow under 15-50 DEG C of scope, and 30
DEG C or 35 DEG C at grow best.
The identification and preservation of bacterial strain
Bacteria total DNA is extracted using TIANGEN DNA extraction kits (Tiangeng biochemical technology, Beijing).It is logical using bacterium
Bacterial strain HW-17 16S rDNA genes are expanded with primer (Lane, 1991).Sent after PCR primer is purified in the innovation of the Wuhan section of holding up
Bio tech ltd is sequenced.The 16S rDNA sequences measured are uploaded in NCBI and compared with known 16S rDNA, are made
With MEGA6 (Tamura et al., 2013) software building systematic evolution tree, its 16S rDNA sequence such as SEQ ID NO.1 institutes
Show.Homology analysis is carried out to sequence, judges that it, for arthrobacterium (Arthrobacter oryzae), is named as HW-17 bacterium
Strain.And the bacterial strain is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University；Postcode 430072,
Preservation date is on March 30th, 2017, and deposit number is：CCTCC NO：M2017156.
The performance test of bacterial strain and analysis
HW-17 bacterial strains provided herein culture 7 days in the culture medium (CMC culture mediums) containing sodium carboxymethylcellulose
During, cellulase activity reaches as high as 18.55U/mL.In the culture medium of this single carbon source, the expression of HW-17 bacterial strains
Cellulase activity is substantially high compared with the cellulase activity (2.80U/mL) that the cellulose degradation strain that other people screen is produced.Cause
This, single for cellulase-producing, HW-17 bacterial strains provided herein have larger application prospect.
The HW-17 bacterial strains that the application is provided can be utilized to be had in cabbage leaf, Rice Leaf, yellow cinnamon leaf, apple skin or weed tree sawdust
Machine thing is grown, and best to the utilizing status of apple skin and cabbage leaf.Therefore, HW-17 bacterial strains can be used as suitable bacterium
Strain is with reluctant fruits and vegetables of degrading, thus this field has huge applications value in fruits and vegetables residue fermenting and producing farm manure.
Specific experiment is as follows：
Apple skins (Apple) of the 1g after liquid nitrogen grinding, Rice Leaf (Rice), yellow cinnamon leaf (Camphor), cabbage are taken respectively
Leaf (Cabbage) or weed tree sawdust (Sawdust) are added into basal medium as carbon source to replace CMC-Na, and control group is to divide
Han not above-mentioned 5 kinds of cellulose samples without liquid nitrogen grinding.Bacterial strain HW-17 is seeded in above-mentioned 10 kinds of culture mediums, 30 DEG C,
D is determined after cultivating 3d under 200r/min600Value and cellulase activity.As a result it is as follows：Bacterial strain HW-17 is in 5 kinds of not ground fibers
Following succession is showed in the culture medium of plain sample:Cabbage leaf>Rice Leaf>Yellow cinnamon leaf>Apple skin>Weed tree sawdust, and 5
Following succession is showed in the culture medium for planting the cellulose sample through liquid nitrogen grinding：Apple skin>Cabbage leaf>Rice Leaf>It is fragrant
Camphor tree leaf>Weed tree sawdust, and the biomass compared with control group is high.In addition, the maximum cellulase activity difference in experimental group and control group
For 4.79UmL-1And 6.69UmL-1, and both of which obtains in the culture medium containing apple skin.Grind the sample of containing cellulose
The contact area of sample and bacterial strain can be increased, so contribute to bacterial strain preferably degraded cellulose.As can be seen here, after grinding
Cellulose sample is more suitable for bacterial strain HW-17 metabolism utilizations, and the cellulase activity produced is higher.
For a large amount of agricultural crop straws produced in agricultural production, such as rice straw, when rice straw and pig manure or chicken
After excrement is mixed with rational ratio, it is inoculated with HW-17 bacterial strains provided herein and ferments 20 into two kinds of cellulose mixture samples
My god, the rice straw in two kinds of biased samples is substantially degraded.In addition, being mixed in pig manure-paddy rice biased sample and chicken manure-paddy rice
Close and maximum cellulase activity is detected in sample for 7.38U/mL and 9.71U/mL.Therefore, HW-17 bacterial strains can be the most suitable
Compost bacterial strain is with agricultural crop straw of degrading, it might even be possible to for the organic farm manure of industrial production.Specific test method is as follows：
The chicken manure (Chicken) or pig manure (Pig) of 20g drying are weighed, is added separately to fill the rice leaf that 20g is ground
(Rice) in conical flask, 1 × 105Sterilize 20min under Pa.Draw 20mL bacterial strain HW-17 seed solutions (D600=1) be seeded to
In above-mentioned sample and mix, be subsequently placed at 30 DEG C and cultivate.Every 2d, leachate is collected, cellulase activity is determined.In addition, will
Leachate carries out gradient dilution, and it is 10 to draw 0.1mL dilution factors-6Dilution, be spread evenly across on CMC-Na solid plates, 30
Cultivated at DEG C and single bacterium colony on flat board is determined after 24h.As a result it is as follows：Bacterial strain HW-17 compost in Rice-Chicken biased samples
After fermentation 12d, cellulase activity maximum (9.71UmL-1)；In Rice-Pig biased samples after fermentation 14d, cellulase activity
Maximum (7.38UmL-1).Cellulase activity variation tendency is consistent with bacterial strain number change trend in sample, and under synchronization
Cellulase activity is above cellulase activity in Rice-Pig biased samples in Rice-Chicken biased samples.Complex microorganism
The rate of change for the cellulase that bacteria fermentation is produced containing chicken manure or pig manure cellulose sample is consistent with the result.In addition, actual see
The chicken manure and rice leaf biased sample measured is degraded more severe compared with pig manure and rice leaf biased sample.Pig manure and paddy rice
Biased sample (C/N=24.7) is high compared with the carbon-nitrogen ratio of chicken manure and the biased sample of paddy rice (C/N=19.7), and the former is more suitable for bacterial strain
HW-17 metabolism.At the initial stage of compost fermentation, bacterial strain HW-17 is preferentially using the organic matter in chicken manure or pig manure to maintain itself
Growth and breeding, during sample nutrition degree to be mixed reduction, bacterial strain HW-17 starts with the cellulose in rice leaf.
<110>：South-Center University For Nationalities
<120>：Arthrobacterium HW-17 bacterial strains of one plant of degraded cellulose and application thereof
<160>：1
<210>：1
<211>：1421
<212>：DNA
<213>：Arthrobacterium（Arthrobacter oryzae）
<400>：1
gggggggcgt ctaaatgcag tcgaacgatg atgcccagct tgctgggtgg attagtggcg 60
aacgggtgag taacacgtga gtaacctgcc cttaactctg ggataagcct gggaaactgg 120
gtctaatacc ggatatgact cctcatcgca tggtgggggg tggaaagctt tttgtggttt 180
tggatggact cgcggcctat cagcttgttg gtgaggtaat ggcttaccaa ggcgacgacg 240
ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc cagactccta 300
cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag cgacgccgcg 360
tgagggatga cggccttcgg gttgtaaacc tctttcagta gggaagaagc gaaagtgacg 420
gtacctgcag aagaagcgcc ggctaactac gtgccagcag ccgcggtaat acgtagggcg 480
caagcgttat ccggaattat tgggcgtaaa gagctcgtag gcggtttgtc gcgtctgccg 540
tgaaagtccg gggctcaact ccggatctgc ggtgggtacg ggcagactag agtgatgtag 600
gggagactgg aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccgatg 660
gcgaaggcag gtctctgggc attaactgac gctgaggagc gaaagcatgg ggagcgaaca 720
ggattagata ccctggtagt ccatgccgta aacgttgggc actaggtgtg ggggacattc 780
cacgttttcc gcgccgtagc taacgcatta agtgccccgc ctggggagta cggccgcaag 840
gctaaaactc aaaggaattg acgggggccc gcacaagcgg cggagcatgc ggattaattc 900
gatgcaacgc gaagaacctt accaaggctt gacatggacc ggaccgggct ggaaacagtc 960
cttccccttt ggggctggtt cacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcaac cctcgttcta tgttgccagc acgtgatggt 1080
ggggactcat aggagactgc cggggtcaac tcggaggaag gtggggacga cgtcaaatca 1140
tcatgcccct tatgtcttgg gcttcacgca tgctacaatg gccggtacaa agggttgcga 1200
tactgtgagg tggagctaat cccaaaaagc cggtctcagt tcggattggg gtctgcaact 1260
cgaccccatg aagtcggagt cgctagtaat cgcagatcag caacgctgcg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcaag tcacgaaagt tggtaacacc cgaagccggt 1380
ggcctaaccc cttgtgggag ggagcttcga agtgacgcat c 1421
Claims (4)
1. arthrobacterium (Arthrobacter oryzae) HW-17 bacterial strains of one plant of degraded cellulose, the bacterial strain is preserved in Chinese allusion quotation
Type culture collection, address：China, Wuhan, Wuhan University；Postcode 430072, preservation date is on March 30th, 2017,
Deposit number is：CCTCC NO：M2017156, the 16S rDNA sequences of the bacterial strain are as shown in SEQ ID NO.1.
2. application of the arthrobacterium HW-17 bacterial strains in degraded cellulose described in claim 1.
3. purposes according to claim 2, it is characterised in that：The arthrobacterium HW-17 bacterial strains are with fowl and animal excrement and agriculture
Crop material is the purposes in raw material production farm manure.
4. purposes according to claim 2, it is characterised in that：The arthrobacterium HW-17 bacterial strains are residual with pericarp, vegetables
Leaf, leaf are the purposes in raw material production farm manure.
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| CN103642722A (en) * | 2013-11-27 | 2014-03-19 | 湖北省烟草公司恩施州公司 | Nicotine-degrading bacterium and application thereof |
| CN104946398A (en) * | 2015-06-26 | 2015-09-30 | 湖北中烟工业有限责任公司 | Method for processing ananas comosus juice through arthrobacter to prepare ananas comosus fragrance for cigarettes |
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| 万文结等: "纤维素降解菌 Arthrobacter oryzae HW-17 的纤维素降解特性及纤维素酶学性质", 《环境科学学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114958672A (en) * | 2022-06-06 | 2022-08-30 | 南京农业大学 | Compound microbial agent for rapid initiation of low-temperature compost as well as preparation method and application thereof |
| CN114958672B (en) * | 2022-06-06 | 2024-03-01 | 南京农业大学 | Low-temperature composting rapid detonation composite microbial agent and preparation method and application thereof |
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