CN113699141A - Slow-release carbon source immobilized aerobic denitrification complex microbial inoculant and preparation method and application thereof - Google Patents
Slow-release carbon source immobilized aerobic denitrification complex microbial inoculant and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a slow-release carbon source immobilized aerobic denitrification composite microbial inoculum and a preparation method and application thereof, wherein the slow-release carbon source immobilized aerobic denitrification composite microbial inoculum is prepared from a composite microbial inoculum and loofah sponge used for immobilizing the composite microbial inoculum, and the composite microbial inoculum has a nitrate nitrogen removal rate of not less than 96%. The invention solves the technical defect of low denitrification efficiency caused by easy flushing of flowing water in the denitrification agent in the prior art by the immobilized aerobic denitrification composite agent. In addition, an aerobic denitrification technology and a plant treatment technology are combined to construct a microorganism-plant remediation system based on the composite microbial inoculum, so that the microbial community of the plant rhizosphere is improved, the plant treatment effect is enhanced, the enhancement effect of the plant on nitrogen pollution removal is further realized, and the deep treatment on drinking water pollution is achieved.
Description
Technical Field
The invention relates to the technical field of environmental biology, in particular to a slow-release carbon source immobilized aerobic denitrification complex microbial inoculum and a preparation method and application thereof.
Background
In recent years, with the development of socio-economy, industrial and agricultural wastewater is continuously discharged into water, so that serious nitrate pollution is caused, and the pollution of drinking water is an important problem. Nitrate pollution can not only cause serious deterioration of water quality such as water eutrophication, but also seriously affect the quality of drinking water. Meanwhile, nitrate can be converted into nitrite in a human body, hemoglobin is directly oxidized into methemoglobin, and finally human tissues are seriously anoxic, so that the human health and the survival of amphibians are influenced. Plant treatment of water pollution as a conventional water treatment technology, is widely applied,
the aerobic denitrification technology can simultaneously remove carbon and nitrogen, and the nitrate is reduced into N by taking a carbon source as an electron donor and nitrate and oxygen as electron acceptors in the denitrification process2Compared with the traditional denitrification, the aerobic denitrification process has the advantages of no oxygen limitation, obvious denitrification efficiency, more economy and the like.
Compared with a powdery microbial inoculum, the free bacterial thallus is easy to wash away by running water, so that the concentration of the aerobic denitrifying bacteria is easy to dilute, the concentration of the thallus is reduced, and the denitrification effect is unstable. The immobilized bacteria can keep the existence of bacteria to a great extent, and solves the problems of low denitrification efficiency, easy loss of strains, poor denitrification stability and the like in the process research of directly putting aerobic denitrifying bacteria. In addition, there is still a need for a microbial-plant integrated treatment system, especially for solving the problem of low nitrate nitrogen removal rate in combined applications.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention prepares the slow-release carbon source immobilized aerobic denitrification composite bacterial agent by compounding the aerobic denitrification strains screened at the early stage and taking the loofah sponge as a carrier. The composite microbial inoculum can solve the technical defect of low denitrification efficiency caused by easy flushing of running water in the denitrification microbial inoculum in the prior art. In addition, the aerobic denitrification technology and the plant treatment technology are combined to construct a microorganism-plant remediation system, so that not only can the microbial community at the rhizosphere of the plant be improved, but also the plant treatment effect can be greatly enhanced, the enhancement effect of the plant on nitrogen pollution removal is realized, and the deep treatment on drinking water pollution is realized. Specifically, the present invention includes the following.
In a first aspect of the invention, a slow-release carbon source immobilized aerobic denitrification complex microbial inoculum is provided, which comprises a complex microbial inoculum and loofah sponge for immobilizing the complex microbial inoculum, and the complex microbial inoculum has nitrate nitrogen removal rate of not less than 96%, such as 97%, 98%, or even 99%.
According to the slow-release carbon source immobilized aerobic denitrification compound bacterial agent disclosed by the invention, preferably, the compound bacterial agent comprises aerobic denitrification bacteria Pseudomonas mendocina LYX and Acidovorax sp.strain YD725, and the Pseudomonas mendocina LYX strain is preserved in China general microbiological culture collection center (CGMCC) for 3-11 days in 2015. The preservation number is CGMCC No.10611, the Acidovorax sp.strain YD725 is preserved in the China center for type culture Collection, and the preservation address is as follows: wuhan, China, the preservation time is No. 5/17 2021, the preservation number is CCTCC NO: m2021536. The isolation and identification of the above strains can be carried out according to methods known in the art.
According to the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum, preferably, the loofah sponge is sodium hydroxide modified loofah sponge. Also preferably, the loofah sponge is modified by 1% -6% of sodium hydroxide. Further preferably, the loofah sponge is modified by 2% -5% of sodium hydroxide.
The slow-release carbon source immobilized aerobic denitrification complex microbial inoculum according to the invention preferably comprises 1:1-2 of Pseudomonas mendocina LYX and Acidovorax sp. 1-1.5, more preferably 1: 1-1.2.
The second aspect of the invention provides a preparation method of a slow-release carbon source immobilized aerobic denitrification complex microbial inoculum, which comprises the following steps:
(1) providing a bacterial suspension containing an aerobic denitrification composite microbial inoculum;
(2) providing a sodium hydroxide modified loofah sponge;
(3) and mixing an organic solution with the bacterial suspension, adding the modified loofah sponge into the obtained mixed solution, and immobilizing the mixed solution and the modified loofah sponge under the condition suitable for immobilization to obtain the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification complex bacterial agent, preferably, the organic solution contains the compound with the structural formula of [ C2H4O]nThe aqueous solution of (4) has a mass-to-volume concentration of 0.05 to 1.2g/mL, preferably 0.06 to 1.1 g/mL.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum, the conditions suitable for immobilization preferably mean that crosslinking is carried out for 12-36h at 0-8 ℃ in the presence of a crosslinking solution, wherein the crosslinking solution is a saturated boric acid solution of 1-10% calcium chloride, and is preferably a saturated boric acid solution of 1-6% calcium chloride.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum, the volume ratio of the organic solution to the bacterial suspension is preferably 1-8:1, and is also preferably 2-6: 1.
In a third aspect of the present invention, there is provided a microbial inoculant-phytoremediation water system comprising an aeration apparatus and a reaction zone, wherein the reaction zone comprises a composite inoculant according to the first aspect and vetiver, also known as Vetiveria zizanioides (L.) as used herein, which is a perennial grass of the family gramineae known as Vetiveria zizanioides (L.) Nash. It will be appreciated by those skilled in the art that the aeration apparatus and reaction zone described above, in combination with other suitable water treatment apparatus, can be used for remediation of contaminated water bodies. Wherein the aeration device can adopt an aeration device known in the field to provide dissolved oxygen for the water body, the reaction zone is used for removing nitrate nitrogen polluting the water body, and the vetiver grass can be fixed by an additional plant fixing device. Preferably, the system further comprises a water inlet, a water outlet and a sampling port.
According to the microbial agent-plant remediation water system, the aeration device preferably comprises an aeration pump, a rotor flow meter and an aeration head, and the aeration head is positioned below the plant fixing device. The water inlet tank is connected with the water inlet through a peristaltic pump and is used for providing a simulated polluted water body. The sample connection includes upper sample connection, middle level sample connection and the lower floor sample connection that from the top down arranges in proper order for handle the sample of water, in order to carry out the treatment effect aassessment of full aspect. And a water outlet is arranged below the lower layer sampling port and used for discharging water.
The microbial inoculant-phytoremediation water system according to the present invention preferably has a nitrate nitrogen removal rate of no less than 96%, such as 97%, 98%, or even 99%.
In a fourth aspect of the invention, the application of the microbial agent-phytoremediation water system according to the third aspect in drinking water source pollution treatment is provided.
The composite microbial inoculum has the effect of synergistically improving the removal efficiency of nitrate nitrogen, and solves the technical defects of repair and treatment of micro-polluted water sources and low denitrification efficiency caused by easy flushing of running water in the denitrification microbial inoculum in the prior art. On one hand, the loofah sponge has wide sources and low price, has porous structure and large specific surface area, is suitable for the growth and attachment of bacteria, has the characteristics of easy storage, stable property, long service life, reusability, high mechanical strength, good stability and porous and rough surface, and is suitable for serving as a carrier of immobilized bacteria. On the other hand, the degradation of the loofah sponge can also be used as a slow-release carbon source to provide a carbon source for the aerobic denitrification process, the aerobic denitrification effect is enhanced, and the loofah sponge is modified by NaOH before the microbial inoculum is prepared, so that a smooth protective film on the loofah sponge falls off, and the aerobic denitrification bacteria can be more easily attached.
In addition, the restoration system combining the aerobic denitrification technology and the plant treatment technology can improve the microbial community at the rhizosphere of the plant, strengthen the plant treatment effect, realize the strengthening effect of the plant on nitrogen pollution removal and achieve the deep treatment on drinking water pollution.
Drawings
Fig. 1 schematically shows a diagram of a microbial-phytoremediation system apparatus according to the present invention.
Fig. 2 shows the nitrate nitrogen removal efficiency of the control group and the enhanced group.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
It is understood by those skilled in the art that other steps or operations can be further included before and after the steps (1) to (3) of preparing the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum, or between any of the steps, for example, the method of the invention is further optimized and/or improved.
Examples
Preparation of microbial inoculum
The selected aerobic denitrifying bacteria are aerobic denitrifying bacteria preserved in a laboratory, namely Pseudomonas mendocina LYX and Acidovorax sp.strain YD725, the strains are respectively preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.10611, China center for type culture Collection with the preservation number of CCTCC NO: m2021536. Before the experiment, the two strains are placed in an LB culture medium to be cultured to a logarithmic phase, the aerobic denitrifying bacteria liquid in the logarithmic phase is centrifuged for 10min at a rotating speed of 8000 r/min, supernatant liquid is poured out, bacteria sediment is left, the two strains are mixed according to a ratio of 1:1, and 0.9% physiological saline is used for fixing the volume to 5mL to form a composite bacteria liquid.
Cutting the loofah sponge into a block body with the thickness of 1cm according to the shape of the loofah sponge, putting the block body into a 4% NaOH solution, boiling for 30min, washing with deionized water to remove the residual NaOH solution, then putting the block body into a 65 ℃ oven, drying for 12h to constant weight, and storing the modified loofah sponge in a dryer.
Weighing 10g of PVA (polyvinyl alcohol), adding 90m of deionized water, uniformly stirring, soaking overnight, placing the PVA solution in a water bath kettle at 90 ℃, heating and continuously stirring until the PVA solution is completely dissolved, taking out the PVA solution, placing the PVA solution at room temperature, cooling the PVA solution to the room temperature, and uniformly mixing 20ml of PVA solution with 5ml of aerobic denitrifying bacteria suspension.
Placing sterilized retinervus Luffae fructus into the mixture, transferring retinervus Luffae fructus soaked in the mixture into 2% calcium chloride saturated boric acid solution (weighing 40g boric acid, adding 20g calcium chloride to constant volume to 1L) with sterile forceps, cross-linking in 4 deg.C refrigerator for 24 hr, washing the formed retinervus Luffae fructus with sterile physiological saline, transferring into 0.9% physiological saline, and storing in 4 deg.C refrigerator for use.
Second, water pollution experiment by using microbial inoculum to strengthen plants
Experimental device and water distribution
The experimental setup is shown in figure 1.
The effective volume of the experimental reactor is 5L, and the experimental reactor comprises a reaction zone, a water inlet, a water outlet, a sampling port, a plant fixing basket and an aeration device, and water is fed in through the creeping of an external water inlet tank.
Water distribution: 0.35g/L of sodium acetate, 0.075g/L of potassium nitrate, 0.1g/L of disodium hydrogen phosphate, 0.1g/L of monopotassium phosphate and 0.01g/L of magnesium sulfate, wherein the concentration of the provided nitrate nitrogen is 10mg/L, and the simulation micro-polluted drinking water environment is realized.
Experimental procedure
The experiment is provided with two groups of reactors which are respectively a control group (without adding microbial inoculum and replaced by a blank carrier) and a strengthening group (with adding microbial inoculum), wherein the two groups of vetiver grass with similar growth tendency and root system condition are used, the experiment is carried out by adding the microbial inoculum with one tenth proportion based on the total volume of blank loofah sponge fillers added in the planting basket, a micropore aeration pump is used for aeration in the running process of the reactors to ensure the dissolved oxygen concentration of the water body, the water body is mixed simultaneously, the hydraulic retention time is controlled at 24h, the fresh water body is replaced every 24h, 5ml of the microbial inoculum is respectively sampled from the upper, middle and lower water taking ports before and after the water body is replaced to obtain 15ml of water sample representing the whole nitrate nitrogen concentration in the water environment, the nitrate nitrogen concentration is measured by using a phenoldisulfonic acid photometry, and the nitrate nitrogen removal rate is calculated. The overall experimental period was 28 days.
As shown in FIG. 2, in the initial stage of microbial inoculum enrichment (stage 1), NO was observed in the control group and the enrichment group3 -The removal of-N was similar, and NO was found in the control and the fortified groups within 1 to 9 days3 -The average N removal rates were 96.82% and 98.03%, respectively, and no difference was observed between the control group and the experimental group at the early stage of the strengtheningThe vetiver probably enters the micro-nutrient water body from the non-nutrient water body, the plants accelerate the absorption of nutrients and are utilized for the growth of the plants, and the strengthening effect of the microbial inoculum cannot be effectively embodied. After day 9 (stage 2), which may be the period in which the plants have spent their thirst for nutrients, the control group had NO3 -The removal rate of-N is reduced from 97.05% on day 9 to 73.73% on day 10 by 23.32%, the average removal rate in the subsequent 10-28 days is only 72.18%, but the enhanced group has a phenomenon opposite to that of the control group, the removal efficiency similar to that in stage 1 is maintained in stage 2, the average removal rate in stage 2 is 97.76%, the phenomenon is caused by the enhanced effect of the microbial inoculum, after the treatment effect of the vetiver is reduced, the microbial inoculum plays a denitrification effect due to the combined effect of the microbial inoculum, the enhanced effect of the system is realized, and the NO of the system is maintained3 -The removal effect of-N ensures that the system has NO3 --a processing efficiency of N.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. The slow-release carbon source immobilized aerobic denitrification composite bacterial agent is characterized by comprising a composite bacterial agent and loofah sponge for fixing the composite bacterial agent, and the composite bacterial agent has nitrate nitrogen removal rate of not less than 96%.
2. The aerobic denitrification complex microbial inoculum for immobilizing the slow-release carbon source as in claim 1, wherein the complex microbial inoculum comprises Pseudomonas mendocina LYX and Acidovorax sp.strain YD725 aerobic denitrification bacteria, wherein the Pseudomonas mendocina LYX strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 10611.
3. The slow-release carbon source immobilized aerobic denitrification complex microbial inoculant according to claim 1, wherein the loofah sponge is sodium hydroxide modified loofah sponge.
4. The slow-release carbon source immobilized aerobic denitrification complex bacterial agent of claim 1, which comprises 1:1-2 of Pseudomonas mendocina LYX and Acidovorax sp.
5. A preparation method of a slow-release carbon source immobilized aerobic denitrification complex microbial inoculum is characterized by comprising the following steps:
(1) providing a bacterial suspension containing an aerobic denitrification composite microbial inoculum;
(2) providing a sodium hydroxide modified loofah sponge;
(3) and mixing an organic solution with the bacterial suspension, adding the modified loofah sponge into the mixed solution, and immobilizing the mixed solution and the modified loofah sponge under the condition suitable for immobilization to obtain the slow-release carbon source immobilized aerobic denitrification composite microbial inoculum.
6. The method for preparing the aerobic denitrification complex microbial inoculum with the immobilized slow-release carbon source as in claim 5, wherein the organic solution contains the compound having the structural formula [ C2H4O]nThe mass-to-volume concentration of the aqueous solution of (1) is 0.05-1 g/mL.
7. The preparation method of the slow-release carbon source immobilized aerobic denitrification complex microbial inoculum is characterized in that the conditions suitable for immobilization are crosslinking for 12-36h at 0-8 ℃ in the presence of a crosslinking solution, and the crosslinking solution is a saturated boric acid solution containing 1-10% of calcium chloride.
8. A microbial inoculant-phytoremediation water system comprising an aeration device and a reaction zone, wherein the reaction zone comprises vetiver and a complex inoculant according to any one of claims 1 to 4.
9. The microbial inoculant-phytoremediation water system of claim 8, wherein the system has a nitrate nitrogen removal rate of no less than 96%.
10. The use of the microbial inoculant-phytoremediation water system of claim 8 in the treatment of pollution of a drinking water source.
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Citations (4)
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|---|---|---|---|---|
| CN105236583A (en) * | 2015-10-28 | 2016-01-13 | 河海大学 | Lotus-root-shaped eutrophic sediment remediation system capable of aeration |
| CN109486706A (en) * | 2018-11-22 | 2019-03-19 | 广东工业大学 | A kind of denitrogenation advantage bacteria agent and its preparation method and application |
| US20190202723A1 (en) * | 2016-06-22 | 2019-07-04 | The Trustees Of Columbia University In The City Of New York | Systems and methods for controlling denitrification in a denitrifying biological reactor |
| CN113373088A (en) * | 2021-06-15 | 2021-09-10 | 广东工业大学 | Aerobic denitrifying bacteria agent with poor nutrition dominance and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105236583A (en) * | 2015-10-28 | 2016-01-13 | 河海大学 | Lotus-root-shaped eutrophic sediment remediation system capable of aeration |
| US20190202723A1 (en) * | 2016-06-22 | 2019-07-04 | The Trustees Of Columbia University In The City Of New York | Systems and methods for controlling denitrification in a denitrifying biological reactor |
| CN109486706A (en) * | 2018-11-22 | 2019-03-19 | 广东工业大学 | A kind of denitrogenation advantage bacteria agent and its preparation method and application |
| CN113373088A (en) * | 2021-06-15 | 2021-09-10 | 广东工业大学 | Aerobic denitrifying bacteria agent with poor nutrition dominance and preparation method and application thereof |
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| 蒋云霞: "水源水库沉积物中好氧反硝化菌群筛选及脱氮特性研究" * |
| 骆坚平 等: "北京典型景观水体好氧反硝化菌组成特征" * |
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