CN110240439B - Preparation method of light high-strength luminous ecological base material solidified by microorganisms - Google Patents
Preparation method of light high-strength luminous ecological base material solidified by microorganisms Download PDFInfo
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Abstract
The invention belongs to the field of ecological slope protection of civil engineering, and particularly discloses a preparation method of a light-weight high-strength luminous ecological substrate solidified by microorganisms, wherein the substrate is prepared from the following raw materials: polypropylene fiber, high impact polystyrene, acrylic resin, bacterial liquid, cementing liquid, luminescent components, raw material soil and water. According to the invention, the polypropylene fiber is added into the soil body to reinforce the soil body, and the high impact polystyrene and the acrylic resin are added, so that the strength of the base material is increased. The base material is formed after the microorganism is solidified, so that the base material has the characteristics of light weight, high strength, no pollution and luminescence, the slope load of the slope can be reduced, and the stability of the slope is improved. The base material meets the requirements of slope ecological protection, can be widely applied to slope protection and landscape design, is light in weight, high in strength and remarkable in landscape effect, and is an ecological base material with wide application range and strong applicability.
Description
Technical Field
The invention relates to the technical field of ecological slope protection of civil engineering, in particular to a preparation method of a light high-strength luminous ecological base material solidified by microorganisms.
Background
The ecological slope protection and slope surface landscape engineering aims at engineering construction and environmental protection, utilizes the anchoring effect of the base material on rock soil to stabilize the slope surface layer and the shallow surface layer and prevent slope surface disasters and water and soil loss, thereby achieving the practical engineering technology for prolonging the service life of the construction engineering, rapidly recovering engineering wounds and improving ecology and beautifying the environment, and becoming an important engineering project for constructing mountainous area traffic, urban construction and real estate development landscape in China. The ecological slope protection technology of China makes great progress, and effectively restrains the problems of water and soil loss, ecological deterioration and slope instability caused by engineering construction. However, the ecological base material often cannot guarantee higher strength requirement in the process of lightening, and has certain limitation in application, and before slope plants grow, the landscape construction of the side slope is difficult, and in addition, ecological protection measures do not have the function of indication and guidance.
The traditional slope protection substrate consumes a large amount of resources, has large workload and poor durability, seriously pollutes the environment in engineering material production engineering, can not plant plants and flowers and plants, is difficult to green and finally destroys the original harmonious ecological environment to a great extent. The microorganism solidified luminous substrate is mainly used for fixing soil and preventing water and soil loss through the chemical effect of microorganism reaction, can be used for landscaping when meeting the requirement of ecological environment, meets the multifunction of slope protection substrate, further improves the microclimate of the surrounding environment where the engineering is located, and restores and improves the ecological environment of the region where the engineering is located.
Disclosure of Invention
Aiming at the defects in the prior art, the inventor thinks that the light luminescent base material solidified by the microorganism can obviously improve the strength characteristic and the physical characteristic, has obvious effect on slope landscape construction and guiding indication, and has wide application range.
The invention mainly aims to increase the strength of a base material and enable the base material to have a luminous characteristic, meet the requirements of slope reinforcement and slope protection and landscape construction, and further provide a preparation method of a light-weight high-strength luminous ecological base material for microbial curing. The method adds polypropylene fibers into the soil to reinforce the soil, and adds high impact polystyrene and acrylic resin to increase the strength of the base material. The base material is formed after the microorganism is solidified, so that the base material has the characteristics of light weight, high strength, no pollution and luminescence, the slope load of the slope can be reduced, and the stability of the slope is improved.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of a light-weight high-strength luminous ecological base material solidified by microorganisms comprises the following specific steps:
(1) preparing a solidified microbial solution 1 and a luminous microbial solution 2;
the immobilized microorganism is Sporosarcina pasteurii (Sporosarcina sp.) and/or Brevibacillus laterosporus (Brevibacillus laterosporus);
the luminescent microorganism is Photobacterium (Photobacterium), Shewanella (Shewanella) and/or Photorhabdus (Photorhabdus);
further, the immobilized microorganism is sporosarcina pasteurii;
further, the luminescent microorganism is photobacterium leiognathi and/or photobacterium brightens;
further, the culture medium for solidifying the microorganisms is NH4+-YE medium; the culture medium of the luminescent microorganism is 2216E liquid culture medium;
the preparation method of the bacterial liquid 1 comprises the following steps: inoculating the immobilized microorganism to NH4+Culturing in YE culture medium for a period of time, and collecting thallus to obtain bacterial liquid 1, wherein the OD600 value of the bacterial liquid 1 is 1.80-2.20;
the preparation method of the bacterial liquid 2 comprises the following steps: inoculating the luminescent microorganism into 2216E liquid culture medium, culturing for a period of time, collecting bacterial liquid, centrifuging, removing supernatant, collecting thalli, taking thalli to redissolve in 0.01mol/L PBS (containing 10mmol/L EDTA and 1mmol/L DTT (dithiothreitol)) with the redissolution ratio of 2.5g of thalli/(20-40) ml PBS to obtain bacterial liquid 2;
(2) preparing a cementing solution;
further, the cementing liquid is composed of peptone, beef extract, glycerol and NaHCO3、MgSO4Urea and Ca (CH)3COO)2And water.
Further, the content of each component in the cementing liquidComprises the following steps: peptone 2.0g/L, beef extract 4.0g/L, glycerin 4.0g/L, NaHCO32.0g/L、MgSO45.0g/L, urea 30.0g/L and Ca (CH)3COO)2 55.0g/L。
(3) Drying the raw material soil in an oven, and sieving the dried raw material soil by a 1mm sieve for later use;
further, the raw material soil is riverway mucky soil.
(4) Sequentially adding polypropylene fibers, high impact polystyrene and acrylic resin into the screened raw material soil in the step (3);
(5) adding water into the mixture obtained in the step (4), and then uniformly stirring in a stirrer;
(6) adding a luminescent component into the mixture obtained in the step (5);
further, the light-emitting component is: firefly luciferase, D-luciferin, ethylenediaminetetraacetic acid (EDTA), and Dithiothreitol (DTT);
firefly luciferase: d-luciferin: EDTA: the weight ratio of DTT is 1: (1-2): (3-4): (1-2);
(7) drip-irrigating the bacterial liquid 1 and the bacterial liquid 2 obtained in the step (1) into the sample obtained in the step (6) in sequence, then soaking the sample in a cementing liquid or spraying the cementing liquid into the sample, and curing for 28 days to obtain a light-weight high-strength luminous ecological base material solidified by microorganisms;
in the actual operation of a construction site, the samples injected with the bacterial liquid are uniformly mixed and sprayed on a side slope, then the samples are sprayed with the cementing liquid, and after multiple spraying, the samples are fully combined to form the light high-strength luminescent substrate.
The dosage of the bacterial liquid 1: and (3) drying and sieving the raw material soil in the step (3), wherein the weight of the dried and sieved raw material soil is 1 mL: (1-6) g;
the amount of the bacterial liquid 2: and (3) drying and sieving the raw material soil in the step (3), wherein the weight of the dried and sieved raw material soil is 1 mL: (3-8) g;
further, the amount of the bacterial liquid 1: and (3) drying and sieving the raw material soil in the step (3), wherein the weight of the dried and sieved raw material soil is 1 mL: (2.5-3.5) g;
the amount of the bacterial liquid 2: and (3) drying and sieving the raw material soil in the step (3), wherein the weight of the dried and sieved raw material soil is 1 mL: (4.5-5.5) g;
further, the stirring speed in the step (5) is set to be 110-125r/min, and the stirring time is 10-20 min.
Further, the polypropylene fiber is of a 9mm grade.
Furthermore, the high impact polystyrene particles are of a grade with the diameter of 2-3 mm.
Furthermore, the acrylic resin particles are of a grade with the diameter of 1-2 mm.
Further, the mixing amount of each component of the base material is (the mass percentage of the raw material soil dried and sieved in the step (3)):
0.2-1 percent of polypropylene fiber, 1-5 percent of high impact polystyrene, 1-3 percent of acrylic resin, 50-70 percent of water and 0.2-0.4 percent of luminescent component.
Since ATP is present in all living cells including microorganisms, in firefly luciferases FL and Mg2+Under the action of (1), D-luciferin LH2Adenylation with ATP is performed to activate the D-luciferin, and the activated D-luciferin is combined with firefly luciferase to form a D-luciferin-AMP complex, and pyrophosphate (PPi) is released. The complex is oxidized by molecular oxygen to form an excited state complex P*-E.AMP giving off CO2Light is emitted when the complex returns from the excited state to the ground state. The reaction formula is as follows:
FL·Ln-AMP+O2→[P*-FL·AMP]+CO2
[P*-FL·AMP]→FL-P+hv+AMP
the polypropylene fiber has the advantages of high strength, good elasticity, wear resistance, corrosion resistance and the like, has stable self property and can not be decomposed by reaction with microorganisms, and can form interfacial force by cementing the generated calcium carbonate precipitates together, thereby effectively inhibiting crack propagation, increasing curing strength, improving the toughness and integrity of the light base material, preventing soil body from being suddenly damaged, providing an adhesion condition for microorganism curing and improving curing effect.
High Impact Polystyrene (HIPS) is a thermoplastic material prepared from elastomer modified polystyrene, has the characteristics of easy processing, rigidity, stable dimension and transparency, and plays a role in toughening and brightening. The material is transparent, light emitted by microorganisms can be emitted through the material, the brightness of the interior of the base material is greatly improved, and the limitation of low impact strength of the material after the microorganisms are cured is also solved.
The acrylic resin has good gloss and color retention, water resistance and corrosion resistance, quick drying, easy construction, excellent fullness, gloss, hardness, solvent resistance and weather resistance, and not only improves the overall brightness of the base material, but also increases the strength of the base material.
Ethylene Diamine Tetraacetic Acid (EDTA) and Dithiothreitol (DTT) can protect active groups of an active center of the enzyme and prevent sulfydryl of firefly luciferase from being oxidized.
Because the permeability coefficient and the water absorption rate are in a negative correlation with the circulating grouting, when the circulating grouting reaches three times or more, the change trend of the curing effect gradually tends to be stable, in addition, the pores of the light base material are more, a good reaction place is provided for the curing of microorganisms, the generated calcium carbonate precipitate can be firmly fixed in the pores of the base material, the internal materials are all cemented together, the strength of the light base material is obviously improved, and the strength of the cured base material far meets the ecological slope fixing requirement.
The technical scheme of the invention is a technology for fixing the reaction of specially selected microorganisms on a selected carrier to ensure that the microorganisms are highly aggregated and keep the biological activity, and can quickly proliferate in large quantities under proper conditions. The technique has the advantages that the adopted bacteria are non-toxic and pollution-free, the environment is not influenced, toxic gas is not discharged in the reaction process, the strength characteristic and the physical characteristic index of the sample are good, and the luminescence performance is excellent. The urease-producing microorganisms catalyze urea hydrolysis to generate calcium carbonate crystals under the condition of a calcium source, partial calcium carbonate products provide media for luminous bacteria, and the luminous bacteria can emit light after chemical reaction. The luminous base material is applied to slope engineering, can be used as scenery spot decoration, constructs various landscape patterns and is used as a guide mark. The base material can be combined with the characteristics of the side slope, the slope surface is designed to be multi-level luminous, the base material is combined with the retaining wall and mutually influenced, the beautifying effect is achieved, ecological disasters can be reduced, the environment is protected, drivers and passengers can conveniently eliminate fatigue, and the appreciation degree is improved. The substrate can also be designed into artistic text form or picture form to display the human character. In summary, in the slope management and landscape design, the method is beneficial to safety protection and can reduce the construction cost, and can generate the comprehensive benefits of ecological landscape, human landscape and landscape aesthetics, and finally can embody the social benefits.
The base material is applied to the field of geotechnical engineering slope protection, and the crack resistance, the impermeability and the mechanical strength of the base material can be obviously improved. The light-emitting base material has wide application prospect in the fields of slope protection, landscape design and the like, has the characteristics of strong adaptability, wide application range and the like, embodies the requirements of characteristics, environmental protection and aesthetic design, and accords with the current concepts of green environmental protection, coordinated development of human and nature and environment.
Compared with the prior art, the invention has the advantages and beneficial effects that:
the invention has the advantages of multifunction of the composite base material, strong applicability, wide application range and the like. When the light high-strength base material is applied to slope protection, the light high-strength base material can play a better role in reinforcement and enclosure, can fully protect the soil body to be stable, is convenient for greening and beneficial to environmental protection, and meanwhile, the base material can be used as a functional decorative material due to self luminescence, thereby having a positive effect on beautifying the environment.
Drawings
FIG. 1 shows the excitation spectrum of the substrate obtained in example 1.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific examples. It will be understood that the examples are for the purpose of further illustrating the subject invention and should not be construed in any way as limiting the scope of the invention.
In the following examples, 9mm grade PPF-9-S-400/10 polypropylene fibers (type: polypropylene fiber monofilament fibers having a length of 9mm, a tensile strength of greater than 400MPa, and an elongation at break of greater than 10%) were purchased from Ruixin cellulose Mill; PH-879 high impact polystyrene of 2-3mm diameter grade was obtained from Shuoguo plastification Co., Ltd; a grade of deglan P24N acrylic resin with a diameter of 1-2mm was obtained from bokupflug engineering, guangzhou; ATCC 11859 Sphaerotheca pasteurii, ATCC 27561 Photobacterium leiognathi and ATCC 11040 Photobacterium brightens are all purchased from Shanghai Lingmei bioengineering Co., Ltd; firefly luciferase (FL, 13.75mg/mL), D-luciferin (Ln, 264364) were purchased from Promega; 2216E liquid culture medium is purchased from QINGDAISHAOBO biotechnology GmbH; EDTA and Dithiothreitol (DTT) were purchased from Sigma; the rest are conventional reagents and instruments.
Example 1:
a light-weight high-strength luminous ecological base material solidified by microorganisms is prepared by the following steps:
(1) preparing a bacterial liquid: inoculation of Sporosarcina pasteurii into NH4+On YE Medium (the Medium contains 20.0g/L of Yeast extract, (NH)4)2SO410.0g/L, adjusting the pH value to 9 with 2g/LNaOH solution), carrying out shaking culture at 25 ℃ for 12h at 140r/min, collecting thalli to obtain a bacterial liquid 1, and measuring the OD600 value of the bacterial liquid to be 2.068 by using an F-2500 fluorescence spectrophotometer; inoculating Photobacterium leiognathi to 2216E liquid culture medium, oscillating at constant temperature of 25 ℃ and 150r/min, culturing for 12h, collecting bacterial liquid, centrifuging at 10000r/min and 4 ℃ for 20min, removing supernatant, collecting thallus, and redissolving in 0.01mol/L pH 7.0PBS (the concentration of EDTA and DTT in the PBS is 10mmol/L and 1mmol/L respectively) at the redissolution ratio of 2.5g thallus/30 ml PBS to obtain bacterial liquid 2;
(2) preparing a cementing liquid: weighing peptone, beef extract, glycerol and NaHCO3、MgSO4Urea and Ca (CH)3COO)2Mixing, adding 1L water, dissolving to desired volume, wherein the final concentration of each material is peptone 2.0g/L, beef extract 4.0g/L, and glycerol 4.0g/L, NaHCO32.0g/L、MgSO45.0g/L and urea 30.0 g-L and Ca (CH)3COO)255.0g/L, stirring and mixing for 18h at the temperature of 30 ℃ and the rotating speed of 200r/min to form cementing liquid;
(3) taking 150g of river sludge raw material soil, drying the river sludge raw material soil in an oven, and screening the dried river sludge raw material soil by using a 1mm sieve for later use;
(4) adding 9mm of polypropylene fiber (the mixing amount is 0.2 percent of the mass of the raw material soil), 2-3mm of high-impact polystyrene particles (the mixing amount is 1 percent of the mass of the raw material soil) and 1-2mm of acrylic resin particles (the mixing amount is 1 percent of the mass of the raw material soil) into the screened raw material soil in the step (3) according to a certain mixing amount ratio in sequence;
(5) adding 90g of water (the mixing amount is 60% of the raw material soil mass) into the mixture obtained in the step (4), and then uniformly stirring in a stirrer (the stirring speed is set to be 110-;
(6) adding 4.77mg of firefly luciferase and 7.21mg of D-luciferin into the mixture obtained in the step (5) in sequence, stirring uniformly, and then adding 16.75mg of Ethylene Diamine Tetraacetic Acid (EDTA) and 6.94mg of Dithiothreitol (DTT) in sequence;
(7) and (4) putting the sample obtained in the step (6) into a substrate die, wherein the die is made of geotextile, and the geotextile is sewn into a cylinder with a diameter of 3.9cm and a height of 8cm and an opening at the upper part, so that the loss of bacteria liquid and nutrient liquid can be delayed, and the curing effect is improved. And (3) slowly dripping 50mL of the bacterial liquid 1 obtained in the step (1) and 30mL of the bacterial liquid 2 into the sample, uniformly stirring, filling the obtained sample by 3 layers, filling about 50g of each layer of the sample into a sample preparation device, and compacting the samples in layers to ensure that the surface of the sample is flat. Soaking the sample injected with the bacterial liquid in 1.5L of cementing liquid, putting the sample into a standard curing box for curing, wherein the curing temperature is (20 +/-2) DEG C, the relative humidity is more than 95 percent, and demolding after curing for 24 hours and continuing curing; and curing to the design age of 28d to obtain the light-weight high-strength luminous ecological base material solidified by the microorganisms.
Example 2:
a microorganism-solidified light-weight high-strength luminous ecological base material, which is prepared by the same method as the example 1 except that no polypropylene fiber is added in the step (4).
Example 3:
a light-weight high-strength luminous ecological base material solidified by microorganisms is prepared by the same method as the embodiment 1 except that photobacterium leiognathi in the step (1) is replaced by photobacterium leiognathi.
Example 4:
a preparation method of a light-weight high-strength luminous ecological base material solidified by microorganisms is the same as that in example 1 except that 9mm polypropylene fibers (the mixing amount is 1% of the mass of raw material soil), 2-3 mm-diameter high-impact polystyrene particles (the mixing amount is 5% of the mass of the raw material soil), and 1-2 mm-diameter acrylic resin particles (the mixing amount is 3% of the mass of the raw material soil) are added in the step (4).
Test example 1:
the light-weight high-strength luminous ecological substrate solidified by the microorganisms prepared in the example 1 is measured by a TU-1810 ultraviolet-visible spectrophotometer to obtain an emission spectrum (lambda) with the emission light of 461nmB) (ii) a The base material obtained in the step (7) in the example 1 is taken out and weighed to about 60g, and an unconfined compressive strength test (refer to ecological slope protection theory and technology) is carried out, so that the soil strength of the base material is 645kPa, and the ecological slope fixing requirement is met.
The emission wavelength was set to 460nm to 462nm, and the excitation wavelength in the range of 220nm to 550nm was scanned, and fluorescence peaks appeared at 232nm, 273nm and 342nm, respectively, as shown in fig. 1. The excitation wavelength is 232nm, and no fluorescence peak exists at 460 nm-462 nm. Wherein 273nm and 342nm are obtained excitation wavelengths, the excitation wavelength is finally determined to be 342nm, and the emission wavelength is 461nm, which shows that the reaction with the D-luciferin can generate fluorescence with the wavelength of 461 nm.
Test example 2:
the light-weight high-strength luminous ecological base material solidified by the microorganisms prepared in the example 2 is measured by a TU-1810 ultraviolet-visible spectrophotometer to obtain an emission spectrum (lambda) with the emission light of 453nmB) (ii) a The base material obtained in the step (7) of the example 2 is taken out and weighed to about 59g, and subjected to an unconfined compressive strength test, so that the soil strength of the base material is 552kPa (the strength is reduced by 14.4% in proportion), and the ecological slope fixing requirement is met.
Test example 3:
the microorganism-cured light-weight high-strength luminous ecological base material prepared in the example 3 is measured by using a TU-1810 ultraviolet-visible spectrophotometerEmission spectrum (lambda) to emission light of 442nmB) (ii) a And (3) taking out the base material obtained in the step (7) in the embodiment 3, weighing about 60g, and performing an unconfined compressive strength test to obtain that the soil body strength of the base material is 618kPa, so that the ecological slope fixing requirement is met.
Test example 4:
the microorganism-cured light-weight high-strength luminous ecological substrate prepared in example 4 is subjected to an emission spectrum (lambda) of 455nm when measured by a TU-1810 ultraviolet-visible spectrophotometerB) (ii) a And (3) taking out the base material obtained in the step (7) in the embodiment 3, weighing about 61g, and performing an unconfined compressive strength test to obtain that the soil strength of the base material is 634kPa, so that the ecological slope fixing requirement is met.
It should be noted that the above-described embodiments may enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. Thus, it will be appreciated by those skilled in the art that the invention may be modified and equivalents may be substituted; all technical solutions and modifications thereof which do not depart from the spirit and technical essence of the present invention should be covered by the scope of the present patent.
Claims (8)
1. A preparation method of a light-weight high-strength luminous ecological base material solidified by microorganisms comprises the following steps:
(1) preparing a solidified microbial solution 1 and a luminous microbial solution 2;
the immobilized microorganism is sporosarcina pasteurii and/or brevibacillus laterosporus;
the luminescent microorganism is photobacterium, Shewanella and/or photorhabdus;
(2) preparing a cementing solution;
the cementing liquid is composed of peptone, beef extract, glycerin and NaHCO3、MgSO4Urea and Ca (CH)3COO）2And water;
(3) drying the raw material soil in an oven, and sieving the dried raw material soil by a 1mm sieve for later use;
(4) sequentially adding polypropylene fibers, high impact polystyrene and acrylic resin into the screened raw material soil in the step (3);
(5) adding water into the mixture obtained in the step (4), and then uniformly stirring;
(6) adding a luminescent component into the mixture obtained in the step (5);
the light-emitting component comprises: firefly luciferase, D-luciferin, ethylenediaminetetraacetic acid and dithiothreitol;
(7) drip-irrigating the bacterial liquid 1 and the bacterial liquid 2 obtained in the step (1) into the sample obtained in the step (6) in sequence, then soaking the sample in a cementing liquid or spraying the cementing liquid into the sample, and curing to obtain a light-weight high-strength luminous ecological base material solidified by microorganisms;
the preparation method of the bacterial liquid 2 comprises the following steps: inoculating the luminescent microorganism into 2216E liquid culture medium, culturing for a period of time, collecting bacterial liquid, centrifuging, removing supernatant, collecting thalli, and taking the thalli to redissolve in PBS (0.01 mol/L, pH = 7.0) containing 10mmol/L EDTA and 1mmol/L DTT, wherein the redissolution ratio is 2.5g of thalli/(20-40) ml of PBS, thus obtaining bacterial liquid 2.
2. The method of claim 1, wherein: the raw material soil is riverway mucky soil.
3. The method of claim 1, wherein: the immobilized microorganism is sporosarcina pasteurii.
4. The method of claim 1, wherein: the luminescent microorganism is photobacterium leiognathi and/or photobacterium brightens.
5. The method of claim 1, wherein:
the preparation method of the bacterial liquid 1 comprises the following steps: inoculating the immobilized microorganism to NH4+Culturing in YE culture medium for a period of time, and collecting thallus to obtain bacterial liquid 1, wherein the OD600 value of the bacterial liquid 1 is 1.80-2.20.
6. The method of claim 1, wherein:
the dosage of the bacterial liquid 1: the weight of the dried and sieved raw material soil in the step (3) is =1 mL: (1-6) g;
the amount of the bacterial liquid 2: the weight of the dried and sieved raw material soil in the step (3) is =1 mL: (3-8) g.
7. The production method according to any one of claims 1 to 6, characterized in that:
the base material comprises the following components in percentage by mass:
0.2-1% of polypropylene fiber, 1-5% of high impact polystyrene, 1-3% of acrylic resin, 50-70% of water and 0.2-0.4% of luminescent component.
8. The production method according to any one of claims 1 to 6, characterized in that:
firefly luciferase: d-luciferin: EDTA: the weight ratio of DTT is 1: (1-2): (3-4): (1-2).
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