CN104560825B - It is a kind of can the husky streptomycete of high-efficiency lignin degrading and its application in the basic conditions - Google Patents
It is a kind of can the husky streptomycete of high-efficiency lignin degrading and its application in the basic conditions Download PDFInfo
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- CN104560825B CN104560825B CN201410856958.0A CN201410856958A CN104560825B CN 104560825 B CN104560825 B CN 104560825B CN 201410856958 A CN201410856958 A CN 201410856958A CN 104560825 B CN104560825 B CN 104560825B
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/26—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof
- C02F2103/28—Nature of the water, waste water, sewage or sludge to be treated from the processing of plants or parts thereof from the paper or cellulose industry
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Abstract
The invention discloses it is a kind of can high-efficiency lignin degrading in the basic conditions novel bacterial：Husky streptomycete (Streptomyces psammoticus) Y 8, the bacterium was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address on 09 02nd, 2014：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number：CGMCC No.9635.Husky streptomycete (Streptomyces psammoticus) Y 8 of the present invention has preferable ligninolytic activity in the basic conditions, to realize that the efficient and inexpensive processing of paper waste lays the foundation.
Description
Technical field：
The invention belongs to environment and field of new energy technologies, and in particular to one kind can in the basic conditions efficient degradation it is wooden
The husky streptomycete of element and its application.
Background technology：
Paper-making industrial waste water is sewage difficult to deal with so far, is also a kind of serious industrial pollution source, the whole world
In the range of, this is considered as public hazards guard key and supervision by every country.Estimate according to united nations environment tissue, whole world paper maker
The waste water that industry is arranged every year is more than more than 27,400,000,000 tons, and there is a middle-size and small-size paper mill of family more than 7000 in China, and annual waste discharge about 21~
2400000000 tons, the total amount of pollutant is 119~1,360,000 tons, and the pollution to environment is only second to chemical industry and metallurgy industry, accounts for industrial wastewater
The 10% of total release, occupies the 3rd, and serious threat is caused to our people's life and ecological safety.
Paper waste is to the harm one of environment main reasons is that lignin and its derivative containing high concentration in waste water
Thing, to the processing of paper waste, one of them topmost problem is exactly to remove these lignin and its derivative.It is main at present
Processing method have：Silvola recovery, acid-precipitation method, bioanalysis etc., wherein Silvola recovery are most ripe during administration of papermaking black liquid is studied
Processing mode, be also the preferable Treatment process of paper mill actual motion at this stage, but Silvola recovery has throwing in application
Money is big, high energy consumption, complex process and to getting the raw materials ready, black liquid extraction, except silicon requirement it is high the shortcomings of, therefore, because Cost Problems are difficult
Received by the especially middle-size and small-size paper mill in paper mill.Acid-precipitation method is simple to operate, by acid adding by the lignin in paper waste
Separate, lignin is reclaimed, using convenience, but need to add a large amount of inorganic acids in operating process, supernatant is in strong
Acidity, subsequent treatment is more difficult to be carried out, treatment cost height and secondary pollution.Relative to conventional at present Silvola recovery and acid-precipitation method
Deng removing the lignin in waste water using Biochemical method paper waste has that cost is low, secondary pollution etc. is not caused to environment
Advantage, therefore with good application prospect.
But the lignin of traditional bioremediation degraded paper waste is there is also some defects, wherein topmost
Due to mainly using alkaline cooking PROCESS FOR TREATMENT raw material in current paper technology, therefore, the pH of paper waste is very high,
Need first to turn down pH value when these black liquor of Biochemical method to adapt to microorganism growth, this considerably increases the complexity of technique
Degree and cost.Therefore, the lignin in paper waste removal waste water is directly handled using the microorganism of alkali resistance, will had non-
Often important application value.The microorganism for the lignin degrading reported at present mainly has whiterot fungi, brown rot fungus, soft-rot fungi etc.,
These microorganisms can under acid or neutrallty condition lignin degrading, but be all difficult to be survived simultaneously under high pH alkalescence condition
And lignin degrading.
The content of the invention：
First purpose of the present invention be to provide it is a kind of can high-efficiency lignin degrading in the basic conditions novel bacterial：Husky chain
Mould (Streptomyces psammoticus) Y-8, the bacterium was preserved in Chinese microorganism strain guarantor on 09 02nd, 2014
Hide administration committee's common micro-organisms center (CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology of institute, deposit number：CGMCC No.9635.
Morphological feature observation to bacterial strain：Gram-positive, bacterium colony is dispersed into lichens shape, and mycelia is in into canescence, spore
Ellipticity, surface is smooth.
Sand streptomycete (Streptomyces psammoticus) Y-8 full genome is extracted with DNA of bacteria extracts kit,
Then using the gene of extraction as template, with universal primer 63f：5’-AGAGTTTGATCCTGGCTCAG-3’；1387r：5’-
ACGGCTACCTTGTTACGACTT-3 ' enters performing PCR amplification, Y-8 16S rDNA sequences such as SEQ ID NO：Shown in 1.It will obtain
Sequence submit NCBI (www.ncbi.nlm.nih.gov) to carry out Megablast, obtain the high correlated series of homology, use
ClustalX softwares are compared, and Mega softwares carry out Phylogenetic Analysis, using adjacent method phylogenetic tree construction.According to
The phylogenetic tree that 16S rDNA sequences are made is as described in Figure 1.Amplification obtains 1.6kbp gene order, in GenBank nucleic acid
Homologous sequence search is carried out in sequence library, Y-8 and Streptomyces psammoticus' (AY999862.1) is homologous
Property be 99%, Y-8 is the new strains of husky streptomycete, is named as husky streptomycete (Streptomyces psammoticus) Y-
Receiving No. 8, GenBank are KP208682.
Research to husky streptomycete (Streptomyces psammoticus) Y-8 shows：In the basic conditions, in 7 days
29.71% is reached to the degradation rate of lignosulfonate, 26.43% is reached to the degradation rate of sulfate-reducing conditions.Show the present invention
Husky streptomycete (Streptomyces psammoticus) Y-8 in the basic conditions have preferable ligninolytic activity.
Therefore, second object of the present invention is to provide husky streptomycete (Streptomyces psammoticus) Y-8 in alkalescence condition
The application of lower lignin degrading.
Described husky streptomycete (Streptomyces psammoticus) Y-8 in the basic conditions lignin degrading should
With specific biodegrading process comprises the following steps：
(1) it is wooden as degrading that husky streptomycete (Streptomyces psammoticus) Y-8, is prepared in the basic conditions
The inoculum of element；
(2), the husky streptomycete (Streptomyces prepared toward addition step (1) in the sample containing lignin
Psammoticus) Y-8 inoculums, shaking table culture, periodically sampling carries out content of lignin detection.
It is preferred that, the preparation method of step (1) is：By husky streptomycete (Streptomyces psammoticus) Y-8 inoculations
In Gause I fluid nutrient medium, 30~40 DEG C, 120~200rpm culture 36h are collected by centrifugation thalline, that is, obtain alkaline bar
The inoculum of lignin degrading under part.
Husky streptomycete (Streptomyces psammoticus) Y-8 prepared by the addition step (1) described in step (2) connects
Thing is planted, inoculum concentration is preferably 5g/L.
The conventional medium that described Gause I fluid nutrient medium is well known to those skilled in the art.
The invention provides a kind of bacterial strain for being capable of high-efficiency lignin degrading in the basic conditions：Husky streptomycete
(Streptomyces psammoticus) Y-8, to realize that the efficient and inexpensive processing of paper waste lays the foundation.
Husky streptomycete (Streptomyces psammoticus) Y-8 of the present invention is on 09 02nd, 2014 is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), address：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica, deposit number：CGMCC No.9635.
Brief description of the drawings：
Fig. 1 is sand streptomycete (Streptomyces psammoticus) Y-8 18S rDNA phylogenetic trees；
Fig. 2 be sand streptomycete (Streptomyces psammoticus) Y-8 under different pH condition to lignin
Degradation rate situation of change；
Fig. 3 is that sand streptomycete (Streptomyces psammoticus) Y-8 degrades wood under the conditions of optimum pH (9.0)
The situation of quality.
Embodiment：
Following examples are that the present invention is further illustrated, rather than limitation of the present invention.
Embodiment 1：
1st, strain isolation
Near the sewage draining exit in paper mill, gather away from pedotheque at earth's surface 10cm, be put into 4 DEG C of refrigerators standby.
The akaline liquid culture medium 100mL containing lignin is prepared, the pedotheque of access 1g collections, 30 after sterilizing cooling
DEG C, shaking table culture under the conditions of 150rpm.Cultured bacterium solution 1mL is taken to be transferred to the fresh akaline liquid training containing lignin after 5 days
Support in base, cultivated under similarity condition.So transfer 3-5 times repeatedly.Then take to be applied to after cultured bacterium solution gradient dilution and contain
On the alkali solid culture medium of lignin, 30 DEG C of quiescent cultures 3-5 days observe colony characteristicses, respectively picking well-grown and bacterium
Fall the different bacterium line culture of form, the line of picking single bacterium colony is cultivated again after 3-5 days, is purified for so repeatedly 3-5 times
Strain.
Purified akaline liquid culture medium of the strain access containing lignin of picking, 30 DEG C, shaking table is trained under the conditions of 150rpm
Support, every 12h samplings once, determination sample content of lignin therein.According to the degraded situation of lignin, further filter out
The strain of high-efficiency lignin degrading in the basic conditions.
Wherein, the component of the akaline liquid culture medium containing lignin is：Lignin 2g/L, glucose 5g/L, NH4NO3
1.5g/L, K2HPO41g/L, KH2PO41.2g/L, MgSO40.2g/L, CaCl20.1g/L, FeSO40.05g/L,
MnSO40.02g/L, distilled water 1000mL, pH value is adjusted as 8~11 using NaOH.Alkali solid culture containing lignin be then
The agar of addition 2% on the basis of fluid nutrient medium component.
2nd, sand streptomycete (Streptomyces psammoticus) Y-8 identification
Observed by morphological feature, the Y-8 that physiological and biochemical property and 16S rDNA gene sequencings are obtained to embodiment 1
Bacterial strain is identified.
(1), morphological feature is observed：Gram-positive, bacterium colony is dispersed into lichens shape, and mycelia is into canescence.Spore is in ellipse
Shape, surface is smooth.
(2), physiological and biochemical property：
(3), 16S rDNA phylogenetic analysis
Extract Y-8's with bacterial genomes DNA extraction kit (being produced by TIANGEN Biotech (Beijing) Co., Ltd.)
Complete genome DNA, then using the genomic DNA of extraction as template, (is had with universal primer by raw work bioengineering (Shanghai) share
Limit company synthesizes) 63f：5’-AGAGTTTGATCCTGGCTCAG-3’；1387r：5 '-ACGGCTACCTTGTTACGACTT-3 ' enter
Performing PCR is expanded.
PCR reaction systems are：The μ L of 1 μ L, 10 × PCR buffer solution of DNA profiling 2, each 1 μ L, Taq DNA of upstream and downstream primer gather
The 2 μ L of μ L, 10 × dNTP of synthase 1, moisturizing to 20 μ L.
PCR amplification programs are：94 DEG C of pre-degeneration 5min；94 DEG C of 40s, 42 DEG C of 1min, 72 DEG C of 1min, 30 circulations, finally
Extend 10min, 4 DEG C of preservations in 72 DEG C.
Amplified production carries out 10g/L agarose gel electrophoresis analyses, purifying and recovery PCR primer, send biotech firm to carry out
Sequencing, Y-8 16S rDNA sequences such as SEQ ID NO：Shown in 1.The sequence of acquisition is submitted into NCBI
(www.ncbi.nlm.nih.gov) Megablast is carried out, the high correlated series of homology is obtained, is carried out with ClustalX softwares
Compare, Mega softwares carry out Phylogenetic Analysis, using adjacent method phylogenetic tree construction.Made according to 16S rDNA sequences
Phylogenetic tree is as shown in Figure 1.
Amplification obtains 1.6kbp gene order, and homologous sequence search, Y- are carried out in GenBank GenBanks
8 and Streptomyces psammoticus (AY999862.1) homology is the new strains that 99%, Y-8 is husky streptomycete,
Husky streptomycete (Streptomyces psammoticus) Y-8 is named as, GenBank receiving number is KP208682.
3rd, the sand degradeds of streptomycete (Streptomyces psammoticus) Y-8 in the basic conditions to lignosulfonate
By husky streptomycete (Streptomyces psammoticus) Y-8 monoclonals bacterium colony of pure culture be seeded to respectively through
In the Gause I fluid nutrient medium for crossing sterilizing, at 30 DEG C, shaken cultivation 36h is carried out under conditions of 150r/min in shaking table,
Then nutrient solution is centrifuged under the conditions of 4000rpm 4min collect thalline, then by weight in wet base 0.5g thalline be seeded to containing
Akaline liquid culture mediums of the 100ml containing lignin carried out in the 250ml conical flasks of biodegradable experiment the biology of lignin
Degradation experiment.The component of akaline liquid culture medium containing lignin is：Lignosulfonate 2g/L, glucose 3g/L, NH4NO3
1.5g/L, K2HPO41g/L, KH2PO41.2g/L, MgSO40.2g/L, CaCl20.1g/L, FeSO40.05g/L,
MnSO40.02g/L, distilled water 1000mL, pH value is adjusted as 10 or so using NaOH.
Conical flask after inoculation is put into shaking table at 30 DEG C, continuous culture 7 days under conditions of 150r/min.In culture
During, a sample was taken every 24 hours, the sample taken daily is centrifuged l0min and removed under 12000g centrifugal force and is hanged
Floating thalline and impurity, determines its absorbance at 280nm, and the lignin for obtaining bacterial strain is calculated according to lignin standard curve
Degradation rate.From the figure 3, it may be seen that the degradation rate of lignosulfonate reaches 29.71% in 7 days.
Degradeds to lignin of husky streptomycete (Streptomyces psammoticus) Y-8 under different pH condition
Rate situation of change is as shown in Figure 2.
4th, the sand drops of streptomycete (Streptomyces psammoticus) Y-8 in the basic conditions to sulfate-reducing conditions
Solution
The Streptomyces psammoticus Y-8 monoclonals bacterium colonies of pure culture are seeded to by sterilizing respectively
In Gause I fluid nutrient medium, at 30 DEG C, shaken cultivation 36h is carried out under conditions of 150r/min in shaking table, then will training
Nutrient solution centrifuges 4min under the conditions of 4000rpm and collects thalline, and then weight in wet base 0.5g thalline is seeded to containing 100ml containing wooden
The akaline liquid culture medium of element carried out in the 250ml conical flasks of biodegradable experiment the biodegradable experiment of lignin.Contain
The component of the akaline liquid culture medium of lignin is：Sulfate-reducing conditions 2g/L, glucose 3g/L, NH4NO31.5g/L,
K2HPO41g/L, KH2PO41.2g/L, MgSO40.2g/L, CaCl20.1g/L, FeSO40.05g/L, MnSO40.02g/L,
Distilled water 1000mL, pH value is adjusted as 10 or so using NaOH.
Conical flask after inoculation is put into shaking table at 30 DEG C, continuous culture 7 days under conditions of 150r/min.In culture
During, a sample was taken every 24 hours, the sample taken daily is centrifuged l0min and removed under 12000g centrifugal force and is hanged
Floating thalline and impurity, determines its absorbance at 280nm, and the lignin for obtaining bacterial strain is calculated according to lignin standard curve
Degradation rate.From the figure 3, it may be seen that the degradation rate of sulfate-reducing conditions reaches 26.43% in 7 days.
Claims (5)
1. a planting sand streptomycete (Streptomyces psammoticus) Y-8, its deposit number is：CGMCC No.9635.
The wood 2. husky streptomycete (Streptomyces psammoticus) Y-8 described in claim 1 degrades in the basic conditions
The application of quality.
3. husky streptomycete (Streptomyces psammoticus) Y-8 according to claim 2 drops in the basic conditions
Solve the application of lignin, it is characterised in that biodegrading process comprises the following steps：
(1) husky streptomycete (Streptomyces psammoticus) Y-8, is prepared in the basic conditions as lignin degrading
Inoculum；
(2), the husky streptomycete (Streptomyces psammoticus) prepared toward addition step (1) in the sample containing lignin
Y-8 inoculums, shaking table culture, periodically sampling carries out content of lignin detection.
4. husky streptomycete (Streptomyces psammoticus) Y-8 according to claim 3 drops in the basic conditions
Solve the application of lignin, it is characterised in that the preparation method of step (1) is：By husky streptomycete (Streptomyces
Psammoticus) Y-8 is inoculated in Gause I fluid nutrient medium, 30~40 DEG C, and 120~200rpm culture 36h, centrifugation is received
Collect thalline, that is, obtain the inoculum of lignin degrading under alkalescence condition.
5. husky streptomycete (Streptomyces psammoticus) Y-8 according to claim 3 drops in the basic conditions
Solve the application of lignin, it is characterised in that husky streptomycete (Streptomyces prepared by the addition step (1) described in step (2)
Psammoticus) Y-8 inoculums, inoculum concentration is 5g/L.
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| 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 |
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| CN102978142A (en) * | 2012-12-13 | 2013-03-20 | 湖南大学 | Rice endophyte (Pantoea sp. Sd-1) for efficiently degrading lignin |
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| CN102978142A (en) * | 2012-12-13 | 2013-03-20 | 湖南大学 | Rice endophyte (Pantoea sp. Sd-1) for efficiently degrading lignin |
Non-Patent Citations (2)
| Title |
|---|
| 曲霉—链霉菌—侧耳筛选及共固定降解碱木素特性;杨玉锁;《中国博士学位论文全文数据库》;20120915;摘要以及第4章4.1及4.3节 * |
| 栗褐链霉菌Streptomyces badius 对木质纤维素的降解研究;黄红丽等;《环境科学与技术》;20060630;4-5,26 * |
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