CN104560825A - Streptomyces psammoticus capable of efficiently degrading lignin under alkaline condition and application thereof - Google Patents

Streptomyces psammoticus capable of efficiently degrading lignin under alkaline condition and application thereof Download PDF

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CN104560825A
CN104560825A CN201410856958.0A CN201410856958A CN104560825A CN 104560825 A CN104560825 A CN 104560825A CN 201410856958 A CN201410856958 A CN 201410856958A CN 104560825 A CN104560825 A CN 104560825A
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husky
streptomycete
streptomyces psammoticus
psammoticus
streptomyces
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CN104560825B (en
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袁振宏
周桂雄
庄新姝
王闻
余强
王琼
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Guangzhou Institute of Energy Conversion of CAS
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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
    • C12R2001/465—Streptomyces
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • 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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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Biodiversity & Conservation Biology (AREA)
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  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)

Abstract

The invention discloses a new strain, namely Streptomyces psammoticus Y-8 capable of efficiently degrading lignin under the alkaline condition. The Streptomyces psammoticus Y-8 is preserved in the CGMCC (China General Microbiological Culture Collection Center) at Sep.2nd in 2014 with the preservation number of CGMCC No.9635, and the address of the CGMCC is Microbiology Research Institute of China Science Academy, No.3 of No.1 yard at Beichen west road of Chaoyang District in Beijing. The Streptomyces psammoticus Y-8, provided by the invention, has better lignin degradation activity under the alkaline condition, and lays the foundation for the efficient and low-cost treatment of paper-making waste water.

Description

A kind of can the husky streptomycete of high-efficiency lignin degrading and application thereof in the basic conditions
Technical field:
The invention belongs to environment and technical field of new energies, be specifically related to a kind of can the husky streptomycete of high-efficiency lignin degrading and application thereof in the basic conditions.
Background technology:
Paper-making industrial waste water is more unmanageable sewage up to now, and be also a kind of serious industrial pollution source, in worldwide, this is all considered as public hazards guard key and supervision by every country.Estimate according to united nations environment tissue, paper industry annual arranged waste water in the whole world is more than more than 27,400,000,000 tons, there is the middle-size and small-size paper mill of family more than 7000 in China, annual waste discharge about 21 ~ 2,400,000,000 tons, the total amount of pollutant is 119 ~ 1,360,000 tons, is only second to chemical industry and metallurgy industry to the pollution of environment, accounts for 10% of trade effluent total release, occupy the 3rd, serious threat is caused to our people's life and ecological safety.
Paper waste harm chief reason to environment is the xylogen and the derivative thereof that contain high density in waste water, removes these xylogen and derivative thereof exactly to one of them topmost problem of process of paper waste.Treatment process main at present has: Silvola recovery, acid-precipitation method, biological process etc., wherein Silvola recovery is processing mode the most ripe in administration of papermaking black liquid research, also be the good Treatment process of present stage paper mill actual motion, but Silvola recovery also exists when applying, and investment is large, energy consumption is high, complex process and to getting the raw materials ready, the shortcoming such as black liquid extraction, silica removal requirement be high, therefore, due to Cost Problems be difficult to receive by especially middle-size and small-size paper mill, paper mill.Acid-precipitation method is simple to operate, by acid adding by the lignin separation in paper waste out, reclaims xylogen, application is convenient, but needs in operating process to add a large amount of mineral acid, and supernatant liquor is strongly-acid, subsequent disposal is more difficult carries out, the high and secondary pollution for the treatment of cost.Relative at present conventional Silvola recovery and acid-precipitation method etc., utilize the Biochemical method paper waste xylogen removed in waste water have cost low, environment is not caused to the advantages such as secondary pollution, therefore there is good application prospect.
But the xylogen of traditional bioremediation degraded paper waste also also exists some defects, wherein topmost is alkaline cooking art breading raw material due to what mainly adopt in current paper technology, therefore, the pH of paper waste is very high, need when these black liquor of Biochemical method first to turn down pH value to adapt to microorganism growth, this considerably increases complexity and the cost of technique.Therefore, utilize the xylogen in the microorganism of alkali resistance direct Processing Paper Wastewater removal waste water, very important using value will be had.The microorganism of the current lignin degrading reported mainly contains whiterot fungi, brown rot fungus, soft-rot bacterium etc., and these microorganisms at lignin degrading under acid or neutrallty condition, but can all be difficult to survival under the alkaline condition of high pH and lignin degrading.
Summary of the invention:
First object of the present invention be to provide a kind of can the novel bacterial of high-efficiency lignin degrading in the basic conditions: husky streptomycete (Streptomyces psammoticus) Y-8, this bacterium was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 09 02nd, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.9635.
Observe the morphological specificity of bacterial strain: Gram-positive, bacterium colony is dispersed into lichens shape, and mycelia becomes canescence, and spore is ellipticity, smooth surface.
Extract test kit by DNA of bacteria and extract the full genome of husky streptomycete (Streptomyces psammoticus) Y-8, then with the gene extracted for template, with universal primer 63f:5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1387r:5 '-ACGGCTACCTTGTTACGACTT-3 ' carries out pcr amplification, and the 16S rDNA sequence of Y-8 is as shown in SEQ ID NO:1.NCBI (www.ncbi.nlm.nih.gov) is submitted to carry out Megablast the sequence obtained, obtain the correlated series that homology is high, compare with ClustalX software, Mega software carries out Phylogenetic Analysis, adopts adjacent method phylogenetic tree construction.The phylogenetic tree done according to 16S rDNA sequence as described in Figure 1.Amplification obtains the gene order of 1.6kbp, homologous sequence search is carried out in GenBank GenBank, the homology of Y-8 and Streptomyces psammoticus (AY999862.1) is 99%, Y-8 is the new strains of husky streptomycete, husky for its called after streptomycete (Streptomyces psammoticus) Y-8, GenBank are accepted number be KP208682.
The research of husky streptomycete (Streptomyces psammoticus) Y-8 is shown: in the basic conditions, in 7 days, 29.71% is reached to the degradation rate of sulfonated lignin, 26.43% is reached to the degradation rate of sulfate-reducing conditions.Show that husky streptomycete of the present invention (Streptomyces psammoticus) Y-8 has good ligninolytic activity in the basic conditions.Therefore, second object of the present invention is to provide the application of husky streptomycete (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions.
The application of described husky streptomycete (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions, concrete degradation method comprises the following steps:
(1) inoculum of husky streptomycete (Streptomyces psammoticus) Y-8 as lignin degrading, is prepared in the basic conditions;
(2), toward containing in the sample of xylogen add husky streptomycete (Streptomyces psammoticus) Y-8 inoculum prepared by step (1), shaking table is cultivated, and content of lignin detection is carried out in regularly sampling.
Preferably, the preparation method of step (1) is: be inoculated in Gause I liquid nutrient medium by husky streptomycete (Streptomyces psammoticus) Y-8,30 ~ 40 DEG C, 120 ~ 200rpm cultivates 36h, collected by centrifugation thalline, namely obtains the inoculum of lignin degrading under alkaline condition.
Add husky streptomycete (Streptomyces psammoticus) Y-8 inoculum prepared by step (1) described in step (2), inoculum size is preferably 5g/L.
The conventional medium that described Gause I liquid nutrient medium is well known to those skilled in the art.
The invention provides a kind of can the bacterial strain of high-efficiency lignin degrading in the basic conditions: husky streptomycete (Streptomyces psammoticus) Y-8, for the process that is efficient and low cost realizing paper waste lays the foundation.
Husky streptomycete of the present invention (Streptomyces psammoticus) Y-8 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 09 02nd, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.9635.
Accompanying drawing illustrates:
Fig. 1 is the 18S rDNA phylogenetic tree of husky streptomycete (Streptomyces psammoticus) Y-8;
Fig. 2 is the degradation rate changing conditions to xylogen of husky streptomycete (Streptomyces psammoticus) Y-8 under different pH condition;
Fig. 3 is the situation of husky streptomycete (Streptomyces psammoticus) Y-8 lignin degrading under optimum pH (9.0) condition.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
1, strains separation
Near the sewage draining exit in paper mill, gather apart from 10cm place, earth's surface pedotheque, put into 4 DEG C of refrigerators for subsequent use.
Preparation is containing the alkaline liquid substratum 100mL of xylogen, and the pedotheque that after sterilizing cooling, access 1g gathers, 30 DEG C, under 150rpm condition, shaking table is cultivated.Get cultured bacterium liquid 1mL after 5 days and proceed to fresh containing in the alkaline liquid substratum of xylogen, cultivate under similarity condition.So repeatedly transfer 3-5 time.Then be applied on the alkali solid substratum containing xylogen after getting cultured bacterium liquid gradient dilution, 30 DEG C of quiescent culture 3-5 days, observe colony characteristics, respectively picking well-grown and the different bacterium of colonial morphology is streak culture, after 3-5 days, picking list bacterium colony is streak culture again, so repeatedly obtains the bacterial classification of purifying for 3-5 time.
The bacterial classification access that picking purifying is good contains the alkaline liquid substratum of xylogen, and 30 DEG C, under 150rpm condition, shaking table is cultivated, every 12h sampling once, and working sample content of lignin wherein.According to the degraded situation of xylogen, filter out the bacterial classification of high-efficiency lignin degrading in the basic conditions further.
Wherein, the component containing the alkaline liquid substratum of xylogen is: xylogen 2g/L, glucose 5g/L, NH 4nO 31.5g/L, K 2hPO 41g/L, KH 2pO 41.2g/L, MgSO 40.2g/L, CaCl 20.1g/L, FeSO 40.05g/L, MnSO 40.02g/L, distilled water 1000mL, with NaOH adjust ph for 8 ~ 11.Alkali solid cultivation containing xylogen is then the agar of interpolation 2% on liquid medium within component basis.
2, the qualification of husky streptomycete (Streptomyces psammoticus) Y-8
By morphological specificity observation, physiological and biochemical property and 16S rDNA gene sequencing, the Y-8 bacterial strain that embodiment 1 obtains is identified.
(1), morphological specificity is observed: Gram-positive, bacterium colony is dispersed into lichens shape, and mycelia becomes canescence.Spore is ellipticity, smooth surface.
(2), physiological and biochemical property:
(3), 16S rDNA phylogenetic analysis
The complete genome DNA of Y-8 is extracted by bacterial genomes DNA extraction kit (being produced by TIANGEN Biotech (Beijing) Co., Ltd.), then with extract genomic dna for template, with universal primer (being synthesized by Sangon Biotech (Shanghai) Co., Ltd.) 63f:5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1387r:5 '-ACGGCTACCTTGTTACGACTT-3 ' carries out pcr amplification.
PCR reaction system is: DNA profiling 1 μ L, 10 × PCR damping fluid 2 μ L, each 1 μ L of upstream and downstream primer, Taq archaeal dna polymerase 1 μ L, 10 × dNTP 2 μ L, moisturizing to 20 μ L.
Pcr amplification program is: 94 DEG C of denaturation 5min; 94 DEG C of 40s, 42 DEG C of 1min, 72 DEG C of 1min, 30 circulations, finally extend 10min in 72 DEG C, 4 DEG C of preservations.
Amplified production carries out the analysis of 10g/L agarose gel electrophoresis, and purifying and recovery PCR primer, send biotech firm to carry out sequencing, the 16S rDNA sequence of Y-8 is as shown in SEQ ID NO:1.NCBI (www.ncbi.nlm.nih.gov) is submitted to carry out Megablast the sequence obtained, obtain the correlated series that homology is high, compare with ClustalX software, Mega software carries out Phylogenetic Analysis, adopts adjacent method phylogenetic tree construction.The phylogenetic tree done according to 16S rDNA sequence as shown in Figure 1.
Amplification obtains the gene order of 1.6kbp, homologous sequence search is carried out in GenBank GenBank, the homology of Y-8 and Streptomyces psammoticus (AY999862.1) is 99%, Y-8 is the new strains of husky streptomycete, husky streptomycete (Streptomyces psammoticus) Y-8, the GenBank acceptance number of called after is KP208682.
3, husky streptomycete (Streptomyces psammoticus) Y-8 is in the basic conditions to the degraded of sulfonated lignin
Husky streptomycete (Streptomyces psammoticus) the Y-8 mono-clonal bacterium colony of pure culture is seeded to respectively in the Gause I liquid nutrient medium of sterilizing, at 30 DEG C, in shaking table, shaking culture 36h is carried out under the condition of 150r/min, then nutrient solution centrifugal 4min under 4000rpm condition is collected thalline, then the thalline of weight in wet base 0.5g is seeded to the biological degradation experiment carrying out carrying out in the 250ml Erlenmeyer flask of biological degradation experiment xylogen containing 100ml containing the alkaline liquid substratum of xylogen.Component containing the alkaline liquid substratum of xylogen is: sulfonated lignin 2g/L, glucose 3g/L, NH 4nO 31.5g/L, K 2hPO 41g/L, KH 2pO 41.2g/L, MgSO 40.2g/L, CaCl 20.1g/L, FeSO 40.05g/L, MnSO 40.02g/L, distilled water 1000mL, with NaOH adjust ph for about 10.
Erlenmeyer flask after inoculation is put into shaking table at 30 DEG C, cultured continuously 7 days under the condition of 150r/min.In culturing process, a sample is got every 24 hours, the sample got every day centrifugal l0min under the centrifugal force of 12000g removes the thalline and impurity that suspend, measures its absorbancy at 280nm place, calculates the Lignin degradation rate of bacterial strain according to xylogen typical curve.As shown in Figure 3, in 7 days, the degradation rate of sulfonated lignin reaches 29.71%.
The degradation rate changing conditions to xylogen of husky streptomycete (Streptomyces psammoticus) Y-8 under different pH condition as shown in Figure 2.
4, husky streptomycete (Streptomyces psammoticus) Y-8 is in the basic conditions to the degraded of sulfate-reducing conditions
The Streptomyces psammoticus Y-8 mono-clonal bacterium colony of pure culture is seeded to respectively in the Gause I liquid nutrient medium of sterilizing, at 30 DEG C, in shaking table, shaking culture 36h is carried out under the condition of 150r/min, then nutrient solution centrifugal 4min under 4000rpm condition is collected thalline, then the thalline of weight in wet base 0.5g is seeded to the biological degradation experiment carrying out carrying out in the 250ml Erlenmeyer flask of biological degradation experiment xylogen containing 100ml containing the alkaline liquid substratum of xylogen.Component containing the alkaline liquid substratum of xylogen is: sulfate-reducing conditions 2g/L, glucose 3g/L, NH 4nO 31.5g/L, K 2hPO 41g/L, KH 2pO 41.2g/L, MgSO 40.2g/L, CaCl 20.1g/L, FeSO 40.05g/L, MnSO 40.02g/L, distilled water 1000mL, with NaOH adjust ph for about 10.
Erlenmeyer flask after inoculation is put into shaking table at 30 DEG C, cultured continuously 7 days under the condition of 150r/min.In culturing process, a sample is got every 24 hours, the sample got every day centrifugal l0min under the centrifugal force of 12000g removes the thalline and impurity that suspend, measures its absorbancy at 280nm place, calculates the Lignin degradation rate of bacterial strain according to xylogen typical curve.As shown in Figure 3, in 7 days, the degradation rate of sulfate-reducing conditions reaches 26.43%.

Claims (5)

1. husky streptomycete (Streptomyces psammoticus) Y-8, its deposit number is: CGMCC No.9635.
2. the application of husky streptomycete according to claim 1 (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions.
3. the application of husky streptomycete according to claim 2 (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions, it is characterized in that, degradation method comprises the following steps:
(1) inoculum of husky streptomycete (Streptomyces psammoticus) Y-8 as lignin degrading, is prepared in the basic conditions;
(2), toward containing in the sample of xylogen add husky streptomycete (Streptomyces psammoticus) Y-8 inoculum prepared by step (1), shaking table is cultivated, and content of lignin detection is carried out in regularly sampling.
4. the application of husky streptomycete according to claim 3 (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions, it is characterized in that, the preparation method of step (1) is: be inoculated in Gause I liquid nutrient medium by husky streptomycete (Streptomyces psammoticus) Y-8,30 ~ 40 DEG C, 120 ~ 200rpm cultivates 36h, collected by centrifugation thalline, namely obtains the inoculum of lignin degrading under alkaline condition.
5. the application of husky streptomycete according to claim 3 (Streptomyces psammoticus) Y-8 lignin degrading in the basic conditions, it is characterized in that, add husky streptomycete (Streptomyces psammoticus) Y-8 inoculum prepared by step (1) described in step (2), inoculum size is 5g/L.
CN201410856958.0A 2014-12-31 2014-12-31 It is a kind of can the husky streptomycete of high-efficiency lignin degrading and its application in the basic conditions Expired - Fee Related CN104560825B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN105132472A (en) * 2015-07-27 2015-12-09 厦门欧米克生物科技有限公司 Usage of streptomyces psammoticus and vanillin production method
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

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Publication number Priority date Publication date Assignee Title
CN105132472A (en) * 2015-07-27 2015-12-09 厦门欧米克生物科技有限公司 Usage of streptomyces psammoticus and vanillin production method
CN105132472B (en) * 2015-07-27 2019-01-08 厦门欧米克生物科技有限公司 The purposes of one seed sand streptomycete and the production method of vanillic aldehyde
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

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