CN104651293A - Compound bacteria with function of synergistically degrading lignocelluloses and organic chlorophenol - Google Patents
Compound bacteria with function of synergistically degrading lignocelluloses and organic chlorophenol Download PDFInfo
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Abstract
The invention discloses compound bacteria with a function of synergistically degrading lignocelluloses and organic chlorophenol, and belongs to the microbial field and the environmental protection field. The compound bacteria which are stable in component characteristic and has a function of synergistically degrading lignocelluloses and organic chlorophenol are obtained by taking filter paper, straws and three organic chlorophenol mixtures as a substrate, and taking oyster mushroom residues, straw mushroom residues and pleurotus eryngii residues as well as different combinations thereof as a culture source through subculture definitive directed education and screening. The bacterial system OEM1 is utilized to process rice straws and organic chlorophenol, and is relatively good in stability and remarkable in degrading effect; the compound bacteria OEM1 can promote the decomposition of lignocelluloses and efficient degradation of organic chlorophenol, and are of great value to expanded application.
Description
Technical field
The invention belongs to microorganism field and field of environment protection, particularly a kind of composite bacteria with lignocellulose and organic chlorophenol Synergistic degradation function.
Background technology
Lignocellulose is the abundantest biomass resource of occurring in nature, by xylogen, Mierocrystalline cellulose and hemicellulose by the interaction such as hydrogen bond and covalent linkage weave in, the lignocellulose structure of height of formation complexity, be difficult to be decomposed by the microorganisms, thus reduce the utilising efficiency of lignocellulosic sources.In order to accelerate decomposition and the utilization of lignocellulose resource, the research team being representative with Cui Zong is when not destroy microorganisms conspiracy relation, adopt long-term restricted outer concentration, filter out the composite bacteria that many groups have stable ligocellulose degradation ability, become thoroughly decomposed speed slowly for solution straw-returning, and the research of utilization promoting lignocellulose material has active effect.
Organic chlorophenol is as the intermediate product (as weaving, papermaking, printing and dyeing etc.) of a kind of important industrial chemicals and industrial link, be widely used in Insecticides (tech) & Herbicides (tech), sanitas and dyestuff intermediate etc., and by spraying, volatilizing, reveal, discharge and the approach entered environment such as burning.For the organic chlorophenol of effective degrade residual in environment, landblink China waits screens domestication and obtains one group and have degraded 4-chlorophenol, 4-nitrophenols and 2 from active sludge, 4, the composite bacteria of 6-trichlorophenol, in the river course that improvement is polluted by aldehydes matter, there is better effects, and Co metabolism effect can be had with the microorganism in environment.
But along with development in science and technology, environmental pollution is more aobvious staggered complexity also, as the solid waste of terminal pollution products, not only contain in raw material the lignocellulose being difficult to degrade, also be enriched the organic pollutant that may remain in the processes such as processing simultaneously, such as paper mill sludge, is not only rich in fiber fines, xylogen and derivative thereof, carbohydrate and salt, and also enrichment concentrates residual lignin in slurrying and form a large amount of organic chlorides in Chlorinated Bleaching process.Organic chloride has typically " three cause " characteristic (teratogenesis, carcinogenic, mutagenesis).Bring external source organic contamination will to soil and crop by directly agricultural for the pollutent (as paper mill sludge etc.) containing organic chloride, bring murder by poisoning to the microorganism of biochemical treatment system.Organic chlorophenol how effectively in degradation of contaminant, the Efficient Conversion simultaneously realizing lignocellulose and organic chlorophenol promotes the key being rich in lignocellulose and organochlorine phenolic comp ' ds pollution (as paper mill sludge etc.) recycling value, and the microbial strains that screening has Synergistic degradation lignocellulose and organic chlorophenol function becomes the key solving foregoing problems.
Summary of the invention
Primary and foremost purpose of the present invention is to overcome the shortcoming that exists in prior art with not enough, provides a kind of preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function.
Another object of the present invention is to provide the composite bacteria with lignocellulose and organic chlorophenol Synergistic degradation function obtained by above-mentioned preparation method.
Object of the present invention is achieved through the following technical solutions: a kind of preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function, comprises the steps:
(1) filter paper and organic chlorophenol storing solution are added in unsterilised liquid medium, add mushroom residue and carry out first stage screening domestication, carry out next generation's switching when fracture and fragmentation appear in filter paper, obtain two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability; The liquid medium interpolation 4g mushroom residue that every 100mL is unsterilised; The addition of screening first stage nutrient solution is 150mL, the liquid medium interpolation 1g filter paper that every 100mL is unsterilised; The unsterilised liquid medium of every 150mL adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of microorganism is 10% (V/V), succeeding transfer culture domestication 30-35 generation;
(2) subordinate phase screening domestication is carried out by the unsterilised liquid medium be transferred to after two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability of screening domestication are blended in step (1) containing rice straw and organic chlorophenol, carry out next generation's switching when rice straw resolves into thread, obtain the composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability; The unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds 0.5g rice straw; The addition that screening subordinate phase contains the unsterilised liquid medium of rice straw and organic chlorophenol is 100mL, and the unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of bacterium liquid is 10% (V/V), succeeding transfer culture domestication 10-15 generation;
In step (1),
Described filter paper is commercially available qualitative filter paper, and being preferably cut into length and width is 6cm × 1cm;
Described organic chlorophenol storing solution is the organic chlorophenol storing solution of mixing of 15% (W/V);
Described organic chlorophenol is preferably ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol, and the mass ratio of ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol is 1:1:1.
Described unsterilised liquid medium is adopted and is prepared with the following method: get peptone 2.5g, Na
2hPO
41.5g, KH
2pO
41.5g, MgCl
26H
2o 0.8g, CaCl
22H
2o 0.8g, yeast extract paste 0.8g, micro-1mL/L, adding distil water is settled to 1000mL, shakes up, and obtains unsterilised liquid medium;
Described trace element is adopted and is prepared with the following method: get MnCl
24H
2o 0.1g, CoCl
26H
2o 0.17g, ZnCl
20.10g, CaCl
20.20g, H
3bO
40.019g, NiCl
26H
2o 0.05g, Na
2moO
42H
2o0.020g, CuSO
45H
2o 0.1g, adding distil water, to 1000mL, by sodium hydroxide adjust ph to neutral, obtains trace element.
Described mushroom residue is the growth matrix abandoned after mushroom is cultivated, and adds in screening system using mushroom residue as the initial bacterial classification source of lignocellulose and organic chlorophenol degradation;
Described mushroom is at least one in flat mushroom, Pleurotus eryngii or straw mushroom.
In step (2),
Described rice straw is preferably adopted and is processed with the following method: after rice straw is soaked 1 day with 1% sodium hydroxide solution, with tap water to neutral, dries, is cut into 0.3-0.5cm long.
There is a composite bacteria OEM1 for lignocellulose and organic chlorophenol degradation capability, obtained by above-mentioned preparation method.
The described composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability preserves with the glycerine of 30%, is placed in-80 DEG C of Refrigerator stores.
Described composite bacteria OEM1 can be applied to lignocellulose degradation and organic chlorophenol.
Preferably, described composite bacteria OEM1 is applied to the substrate that degraded contains 0.5-4.0% lignocellulose and the organic chlorophenol of 50-75mg/L.
The condition optimization of described degraded is in 28 DEG C, 120r/min, degraded 12d.
When described composite bacteria OEM1 is applied to lignocellulose degradation and organic chlorophenol, activated for two generations, during degraded, the inoculum size of composite bacteria OEM1 is 10% (V/V).
The present invention has following advantage and effect relative to prior art:
(1) the present invention with lignocellulose and organic chlorophenol for target is degraded substrate, mushroom residue is bacterial classification source, the composite bacteria OEM1 that one group has efficient degradation lignocellulose and organic chlorophenol function under normal-temperature aerobic condition has been constructed in screening, and by degrading rice straw and the test of organic chlorophenol, verify the lignocellulose degradation of this composite bacteria and the usefulness of organic chlorophenol and functional stabilization.
(2) the present invention is directed to the lignocellulose that current industrial solid wastes (as paper mill sludge etc.) not only contain remaining difficult degradation in raw materials for production, also be enriched the organic chloride that may remain in the processes such as processing, how residual in efficient degradation solid waste lignocellulose and organic chloride become the key of its recycling simultaneously.The invention provides the approach solving and be rich in lignocellulose and organic chloride environmental pollution and realize recycling key issue, achieve lignocellulose and the efficient Synergistic degradation of organic chloride, there is the new lignocellulose of exploitation and organic chloride high-efficiency degradation composite bacteria resource, promote the development of microbial ecology, prevent trade waste containing lignocellulose or organic chloride as the environmental pollution of paper mill sludge and the significance improving its resource utilization rate.
(3) screening process of the present invention is simple, workable; The composite bacteria OEM1 composition obtained and stable in properties, can efficient degradation Mierocrystalline cellulose, hemicellulose and xylogen, the simultaneously organic chlorophenol of effective degraded.
Accompanying drawing explanation
Fig. 1 is the degradation results of composite bacteria OEM1 to rice straw of embodiment 2;
Fig. 2 is that the composite bacteria OEM1 of embodiment 2 is to the degradation results of Mierocrystalline cellulose, hemicellulose and xylogen;
Fig. 3 is that the composite bacteria OEM1 of embodiment 2 is to the degradation results of organic chlorophenol;
Fig. 4 is pH value result of variations in the composite bacteria OEM1 lignocellulose degradation of embodiment 2 and organic chlorophenol process.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
There is a preparation method for the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function, comprise the steps:
(1) filter paper and organic chlorophenol storing solution are added in unsterilised liquid medium, add mushroom residue and carry out first stage screening domestication, carry out next generation's switching when obviously fracture and fragmentation appear in filter paper, obtain two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability; The liquid medium interpolation 4g mushroom residue that every 100mL is unsterilised; The addition of the liquid medium that the screening first stage is unsterilised is 150mL, the liquid medium interpolation 1g filter paper that every 100mL is unsterilised; The unsterilised liquid medium of every 150mL adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of microorganism is 10% (V/V), succeeding transfer culture domestication 30-35 generation;
(2) subordinate phase screening domestication is carried out by the unsterilised liquid medium be transferred to after two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability of screening domestication are blended in step (1) containing rice straw and organic chlorophenol, carry out next generation's switching when rice straw resolves into thread, obtain the composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability; The unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds 0.5g rice straw; The addition that screening subordinate phase contains the unsterilised liquid medium of rice straw and organic chlorophenol is 100mL, and the unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of bacterium liquid is 10% (V/V), succeeding transfer culture domestication 10-15 generation;
In step (1),
Described filter paper is commercially available qualitative filter paper, and being cut into length and width is 6cm × 1cm;
Described organic chlorophenol storing solution is the organic chlorophenol storing solution of mixing of 15% (W/V);
Described organic chlorophenol is ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol, and the mass ratio of ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol is 1:1:1.
Described unsterilised liquid medium is adopted and is prepared with the following method: get peptone 2.5g, Na
2hPO
41.5g, KH
2pO
41.5g, MgCl
26H
2o 0.8g, CaCl
22H
2o 0.8g, yeast extract paste 0.8g, micro-1mL/L, adding distil water is settled to 1000mL, shakes up, and obtains unsterilised liquid medium;
Described trace element is adopted and is prepared with the following method: get MnCl
24H
2o 0.1g, CoCl
26H
2o 0.17g, ZnCl
20.10g, CaCl
20.20g, H
3bO
40.019g, NiCl
26H
2o 0.05g, Na
2moO
42H
2o0.020g, CuSO
45H
2o 0.1g, adding distil water, to 1000mL, by sodium hydroxide adjust ph to neutral, obtains trace element.
Described mushroom residue is the growth matrix abandoned after mushroom is cultivated, and adds in screening system using mushroom residue as the initial bacterial classification source of lignocellulose and organic chlorophenol degradation;
Described mushroom is at least one in flat mushroom, Pleurotus eryngii or straw mushroom.
In step (2),
Described rice straw is adopted and is processed with the following method: after rice straw is soaked 1 day with 1% sodium hydroxide solution, with tap water to neutral, dries, is cut into 0.3-0.5cm long.
Embodiment 2
Adopt the composite bacteria OEM1 Treating straw with lignocellulose and organic chlorophenol degradation capability and the ortho chloro phenol, 2 of embodiment 1,4-Dichlorophenol and 2,4,6-trichlorophenol, analyzes composite bacteria OEM1 to lignocellulose and the degradation characteristic of organic chlorophenol and the stability of degeneration system.
(1) the composite bacteria OEM1 at-80 DEG C of freezen protective is taken out activation two generation, shaking culture under 28 DEG C and 120r/min condition, first-generation activation 3-4d, switching activation s-generation activation 5-6d.
(2) by the rice straw of taking from certain agriculture university experimental plot with 1% soaking with sodium hydroxide after 1 day, with tap water to neutral, dry, to be cut into 0.3-0.5cm long.
(3) by peptone 2.5g, Na
2hPO
41.5g, KH
2pO
41.5g, MgCl
26H
2o 0.8g, CaCl
22H
2o 0.8g, yeast extract paste 0.8g, micro-1mL/L, adding distil water 1000mL constant volume, obtains unsterilised liquid medium.(trace element: MnCl
24H
2o 0.1g, CoCl
26H
2o 0.17g, ZnCl
20.10g, CaCl
20.20g, H
3bO
40.019g, NiCl
26H
2o 0.05g, Na
2moO
42H
2o0.020g, CuSO
45H
2o 0.1g, adding distil water is to 1000mL, extremely neutral by sodium hydroxide adjust ph.)。
(4) ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol are dissolved in ethanol according to mass ratio 1:1:1, are configured to organic chlorophenol storing solution that respective concentration is 5% (W/V);
(5) rice straw in step (2) is added in the unsterilised liquid medium of 90mL, at 121 DEG C of sterilizing 20min; The weight of rice straw and the volume ratio of unsterilised liquid medium are 0.5% (W/V);
(6) organic chlorophenol storing solution of 50 μ L is added to step (3) in the unsterilised liquid medium of cooling;
(7) the composite bacteria OEM1 of activation in step (1) is added in the organic chlorophenol storing solution in step (4), inoculum size is with 10% (V/V), 28 DEG C, 120r/min shaking culture 12 days, measure that pH in degradation process, stalk are weightless, ligocellulose degradation leads and the index such as organochlorine Phenol degradation rate, result as Figure 1-4.
As can be seen from Fig. 1-4, after composite bacteria OEM1 process 12d, straw degradative 54.43%, the degradation rate of Mierocrystalline cellulose, hemicellulose and xylogen reaches 86.84%, 91.22% and 42.74% respectively.Organic chlorophenol has substantially been degraded in 9d, and the degradation rate of ortho chloro phenol, 2,4-Dichlorophenols and 2,4,6-trichlorophenol is respectively 100%, 100% and 82.88%.Test-results shows, composite bacteria OEM1 has good lignocellulose and organic chlorophenol Synergistic degradation ability.In degradation process, the Acid-Base System of degeneration system keeps stable, and pH value, by initial 5.5, rises to 7.45 at front 3d rapidly, after this maintains 7.5.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. there is a preparation method for the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function, it is characterized in that, comprise the steps:
(1) filter paper and organic chlorophenol storing solution are added in unsterilised liquid medium, add mushroom residue and carry out first stage screening domestication, carry out next generation's switching when fracture and fragmentation appear in filter paper, obtain two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability; The liquid medium interpolation 4g mushroom residue that every 100mL is unsterilised; The addition of screening first stage nutrient solution is 150mL, the liquid medium interpolation 1g filter paper that every 100mL is unsterilised; The unsterilised liquid medium of every 150mL adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of microorganism is 10%, succeeding transfer culture domestication 30-35 generation;
(2) subordinate phase screening domestication is carried out by the unsterilised liquid medium be transferred to after two groups of composite bacteria with Mierocrystalline cellulose and organic chlorophenol degradation capability of screening domestication are blended in step (1) containing rice straw and organic chlorophenol, carry out next generation's switching when rice straw resolves into thread, obtain the composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability; The unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds 0.5g rice straw; The addition that screening subordinate phase contains the unsterilised liquid medium of rice straw and organic chlorophenol is 100mL, and the unsterilised liquid medium that every 100mL contains rice straw and organic chlorophenol adds the organic chlorophenol storing solution of 50 μ L; In screening domestication process, the inoculum size of bacterium liquid is 10%, succeeding transfer culture domestication 10-15 generation.
2. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 1, is characterized in that, the organic chlorophenol storing solution described in step (1) is the organic chlorophenol storing solution of 15% mixing.
3. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 2, it is characterized in that, described organic chlorophenol is ortho chloro phenol, 2,4-Dichlorophenol and 2,4,6-trichlorophenol, ortho chloro phenol, 2, the mass ratio of 4-Dichlorophenol and 2,4,6-trichlorophenol is 1:1:1.
4. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 1, it is characterized in that, unsterilised liquid medium described in step (1) is adopted and is prepared with the following method: get peptone 2.5g, Na
2hPO
41.5g, KH
2pO
41.5g, MgCl
26H
2o 0.8g, CaCl
22H
2o 0.8g, yeast extract paste 0.8g, micro-1mL/L, adding distil water is settled to 1000mL, shakes up, and obtains unsterilised liquid medium.
5. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 4, is characterized in that, described trace element is adopted and prepared with the following method: get MnCl
24H
2o0.1g, CoCl
26H
2o 0.17g, ZnCl
20.10g, CaCl
20.20g, H
3bO
40.019g, NiCl
26H
2o0.05g, Na
2moO
42H
2o 0.020g, CuSO
45H
2o 0.1g, adding distil water, to 1000mL, by sodium hydroxide adjust ph to neutral, obtains trace element.
6. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 1, is characterized in that, the mushroom residue described in step (1) is the growth matrix abandoned after mushroom is cultivated.
7. the preparation method with the composite bacteria of lignocellulose and organic chlorophenol Synergistic degradation function according to claim 1, it is characterized in that, rice straw described in step (2) is adopted and is processed with the following method: after rice straw is soaked 1 day with 1% sodium hydroxide solution, with tap water to neutral, dry, be cut into 0.3-0.5cm long.
8. there is a composite bacteria OEM1 for lignocellulose and organic chlorophenol degradation capability, obtained by the preparation method described in any one of claim 1-7.
9. the composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability according to claim 8 is applied to lignocellulose degradation and organic chlorophenol.
10. the application with the composite bacteria OEM1 of lignocellulose and organic chlorophenol degradation capability according to claim 9, it is characterized in that, the described composite bacteria OEM1 with lignocellulose and organic chlorophenol degradation capability is applied to the substrate that degraded contains 0.5-4.0% lignocellulose and the organic chlorophenol of 50-75mg/L; The condition of described degraded is in 28 DEG C, 120r/min, degraded 12d; The described inoculum size with the composite bacteria OEM1 of lignocellulose and organic chlorophenol degradation capability is 10%.
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| CN115466704A (en) * | 2022-11-02 | 2022-12-13 | 中国农业科学院农业环境与可持续发展研究所 | Method for constructing lactic acid-acetic acid-producing microbial flora under non-sterile condition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105524835A (en) * | 2016-01-07 | 2016-04-27 | 华南农业大学 | Obtaining method and application of salt-tolerant cellulose decomposition bacteria colony |
| CN110819540A (en) * | 2019-12-04 | 2020-02-21 | 刘梦婧 | Preparation method of biomass decomposing bacteria |
| CN115466704A (en) * | 2022-11-02 | 2022-12-13 | 中国农业科学院农业环境与可持续发展研究所 | Method for constructing lactic acid-acetic acid-producing microbial flora under non-sterile condition |
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