CN113699141B - Sustained-release carbon source immobilized aerobic denitrification composite microbial inoculant and preparation method and application thereof - Google Patents

Sustained-release carbon source immobilized aerobic denitrification composite microbial inoculant and preparation method and application thereof Download PDF

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CN113699141B
CN113699141B CN202110990415.8A CN202110990415A CN113699141B CN 113699141 B CN113699141 B CN 113699141B CN 202110990415 A CN202110990415 A CN 202110990415A CN 113699141 B CN113699141 B CN 113699141B
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aerobic denitrification
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CN113699141A (en
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许燕滨
杨德
江进
李宁
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Guangdong University of Technology
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Abstract

The invention discloses a slow-release carbon source immobilized aerobic denitrification compound microbial inoculant, a preparation method and application thereof, wherein the slow-release carbon source immobilized aerobic denitrification compound microbial inoculant is prepared from a compound microbial inoculant and loofah sponge for immobilizing the compound microbial inoculant, and the compound microbial inoculant 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 the denitrification microbial inoculum by running water in the prior art by the immobilized aerobic denitrification compound microbial inoculum. In addition, the aerobic denitrification technology and the plant treatment technology are combined to construct a microorganism-plant restoration system based on the composite microbial agent, so that not only is the plant rhizosphere microorganism community improved and the plant treatment effect enhanced, but also the enhancement of the nitrogen pollution removal of plants is further realized, and the advanced treatment of drinking water pollution is achieved.

Description

Sustained-release carbon source immobilized aerobic denitrification composite microbial inoculant and preparation method and application thereof
Technical Field
The invention relates to the technical field of environmental biology, in particular to a slow-release carbon source immobilized aerobic denitrification compound microbial inoculant, a preparation method and application thereof.
Background
In recent years, with the development of social and economic technologies, industrial and agricultural wastewater is continuously discharged into water, so that serious nitrate pollution is caused, and the pollution is an important problem facing drinking water pollution. The nitrate pollution not only can cause serious deterioration of water quality such as water eutrophication and the like, but also seriously affects the quality of drinking water. Meanwhile, nitrate can be converted into nitrite in a human body, hemoglobin is directly oxidized into methemoglobin, and finally serious hypoxia of human tissues is caused, so that the human health and the survival of amphibians are affected. The plant treatment water pollution is widely applied as a conventional water treatment technology,
the aerobic denitrification technology can remove carbon and nitrogen simultaneously, and in the denitrification process, a carbon source is used as an electron donor, nitrate and oxygen are used as an electron acceptor, and nitrate is reduced into N 2 Compared with the traditional denitrification, the aerobic denitrification process has the advantages of no limitation of oxygen, obvious denitrification efficiency, more economy and the like.
Compared with the powder 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 microbial inoculum can maintain the existence of thalli to a great extent, and solves the problems of low denitrification efficiency, easy strain loss, poor denitrification stability and the like existing in the process research of directly putting aerobic denitrifying bacteria into the process research to a certain extent. In addition, a microorganism-plant comprehensive treatment system is still needed at present, and particularly, the problem that the nitrate nitrogen removal rate is not high in combined application can be solved.
Disclosure of Invention
In order to solve the technical problems in the prior art, the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum is prepared by compounding the aerobic denitrification strain screened in the earlier stage and taking the loofah sponge as a carrier. The composite microbial inoculum disclosed by the invention can be used for solving the technical defect of low denitrification efficiency caused by easy flushing of running water in the prior art. In addition, the aerobic denitrification technology and the plant treatment technology are combined to construct a microorganism-plant repair system, so that not only can plant rhizosphere microorganism communities be improved, but also plant treatment effect can be greatly enhanced, strengthening effect of plants on nitrogen pollution removal is realized, and deep treatment of drinking water pollution is achieved. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided a slow-release carbon source immobilized aerobic denitrification complex microbial inoculant comprising a complex microbial inoculant and retinervus luffae fructus for immobilizing the complex microbial inoculant, wherein the complex microbial inoculant has a nitrate nitrogen removal rate of not less than 96%, for example 97%, 98%, or even 99%.
The slow-release carbon source immobilized aerobic denitrification composite microbial agent according to the invention preferably comprises aerobic denitrification bacteria Arthrobacter sp YC-RL1 andAcidovorax sp.strain YD725, arthrobacter bacteria (Arthrobacter sp.) YC-RL1 are deposited in the China general microbiological culture collection center CGMCC with a deposition time of 2015 for 3 months and 11 days. The preservation number is CGMCC No.10611,Acidovorax sp.the strain YD725 is preserved in China Center for Type Culture Collection (CCTCCM) with the preservation time of 2021, 5 months and 17, and the preservation number of CCTCCM 2021536. The isolation and identification methods of the above strains can be performed according to methods known in the art.
According to the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum disclosed by the invention, preferably, the loofah sponge is sodium hydroxide modified loofah sponge. It is also preferable that the modification treatment of the retinervus Luffae fructus is performed by 1% -6% sodium hydroxide. Further preferably, the modification treatment of the retinervus Luffae fructus is performed by 2% -5% sodium hydroxide.
The slow-release carbon source immobilized aerobic denitrification composite microbial agent according to the invention preferably comprises 1:1-2 of Arthrobacter bacteria (Arthrobacter sp.) YC-RL1 and by weight of the composite microbial agentAcidovorax sp.strain YD725, also preferably 1:1-1.5, further preferably 1:1-1.2.
The invention provides a preparation method of a slow-release carbon source immobilized aerobic denitrification compound microbial inoculant, which comprises the following steps:
(1) Providing a bacterial suspension containing an aerobic denitrification compound bacterial agent;
(2) Providing sodium hydroxide modified loofah sponge;
(3) And mixing an organic solution with the bacterial suspension, adding the modified loofah into the obtained mixed solution, and immobilizing the mixed solution and the modified loofah under the condition suitable for immobilization to obtain the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum, preferably, the organic solution contains a compound having a structural formula of [ C ] 2 H 4 O] n The mass volume ratio concentration of the aqueous solution is 0.05-1.2g/mL, and preferably 0.06-1.1g/mL.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification composite microbial inoculum, preferably, the condition suitable for immobilization refers to crosslinking 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% of calcium chloride, and also preferably a saturated boric acid solution of 1-6% of calcium chloride.
According to the preparation method of the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum, the volume ratio of the organic solution to the microbial suspension is preferably 1-8:1, and is also preferably 2-6:1.
In a third aspect the present invention provides 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 referred to herein as vetiver, referred to as vetiver under the discipline of the subject matterVetiveria zizanioides(l.) Nash grass perennial herb. It will be appreciated by those skilled in the art that the aeration device and reaction zone described above in combination with other suitable water treatment devices can be used for the remediation of contaminated water. Wherein the aeration device can employ aeration devices known in the art to provide dissolved oxygen in the water, the reaction zone is used for nitrate nitrogen removal of the contaminated water, and vetiver can be immobilized by another plant immobilization device. Preferably, the system further comprises a water inlet, a water outlet, a sampling port.
The microbial agent-phytoremediation water system according to the present invention preferably comprises an aeration pump, a rotameter and an aeration head, the aeration head being located below the plant fixture. The water inlet tank is connected with the water inlet through a peristaltic pump and is used for providing simulated polluted water. The sampling port comprises an upper layer sampling port, a middle layer sampling port and a lower layer sampling port which are sequentially arranged from top to bottom, and is used for processing water body sampling so as to perform comprehensive treatment effect evaluation. And a water outlet is arranged below the lower sampling port and is used for removing water.
The microbial inoculant-phytoremediation water system according to the present invention preferably has a nitrate nitrogen removal of no less than 96%, such as 97%, 98%, or even 99%.
In a fourth aspect of the invention, there is provided the use of a microbial inoculant-phytoremediation water system according to the third aspect in the treatment of pollution of a drinking water source.
The composite microbial inoculum has the effect of synergistically improving the nitrate nitrogen removal efficiency, and solves the technical defect of low denitrification efficiency caused by easy flushing of running water in the prior art of remediation and treatment of micro-polluted water sources. On one hand, the loofah sponge has the characteristics of wide sources, low price, porous structure, large specific surface area, suitability for the growth and attachment of bacteria, easiness in storage, stable property, long service life and reusability, and is high in mechanical strength, good in stability, rough in surface porosity and 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, so that the aerobic denitrification effect is enhanced, and the loofah sponge is modified by NaOH before the preparation of the microbial inoculum, so that a smooth protective film on the loofah sponge is fallen off, and the attachment of aerobic denitrification bacteria is easier.
In addition, the repair system combining the aerobic denitrification technology and the plant treatment technology can improve plant rhizosphere microbial communities, strengthen plant treatment effects, realize strengthening effect on nitrogen pollution removal of plants and achieve advanced treatment on drinking water pollution.
Drawings
FIG. 1 schematically shows a microbial-phytoremediation system according to the present invention.
FIG. 2 is a graph of nitrate nitrogen removal efficiency for the control and enhanced groups.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 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 will be appreciated by those skilled in the art that other steps or operations may be included before or after the steps (1) - (3) of preparing the slow-release carbon source immobilized aerobic denitrification complex microbial agent, or between any of these steps, for example, further optimizing and/or improving the methods of the present invention.
Examples
1. Preparation of microbial inoculum
The selected aerobic denitrifying bacteria are laboratory preserved aerobic denitrifying bacteria Arthrobacter sp YC-RL1 andAcidovorax sp.strain YD725 is preserved in China general microbiological culture collection center CGMCC with the preservation number of CGMCC No.10611 and China center for type culture collection with the preservation number of CCTCCM 2021536. Before the experimentCulturing the two strains in LB culture medium to logarithmic phase, centrifuging the aerobic denitrifying bacteria liquid in logarithmic phase at 8000 r/min for 10 min, decanting supernatant to leave bacterial precipitate, mixing the two bacterial precipitate at a ratio of 1:1, and fixing volume to 5 mL with 0.9% physiological saline to form composite bacteria liquid.
Cutting retinervus Luffae fructus into 1cm thick blocks according to its shape, boiling in 4% NaOH solution for 30min, washing with deionized water to remove residual NaOH solution, oven drying at 65deg.C for 12 h to constant weight, and storing in a dryer.
10g of PVA (polyvinyl alcohol) is weighed, added with 90m of deionized water, evenly stirred and soaked overnight, the PVA solution is placed in a water bath kettle at 90 ℃ and is heated and continuously stirred until the PVA solution is completely dissolved, the PVA solution is taken out and cooled to room temperature, and 20 ml of PVA solution and 5 ml aerobic denitrifying bacteria bacterial suspension are evenly mixed.
Placing sterilized retinervus Luffae fructus into the mixed solution, allowing the mixed solution to fully enter into gap of retinervus Luffae fructus, transferring retinervus Luffae fructus soaked in the mixed solution into saturated boric acid solution containing 2% calcium chloride (40 g boric acid is weighed, 20g calcium chloride is added to constant volume of 1L), placing into refrigerator at 4deg.C for crosslinking 24 h, washing the formed retinervus Luffae fructus with sterile physiological saline, transferring into 0.9% physiological saline, and storing in refrigerator at 4deg.C for use.
2. Experiment for restoring water pollution by using bacteria agent reinforced plants
(1) Experimental device and water distribution
Experimental setup 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 peristaltic water inflow is realized through an external water inlet tank.
And (3) water distribution: sodium acetate 0.35 g/L, potassium nitrate 0.075 g/L, disodium hydrogen phosphate 0.1 g/L, potassium dihydrogen phosphate 0.1 g/L, magnesium sulfate 0.01 g/L, and the nitrate nitrogen concentration provided is 10 mg/L, simulating a slightly polluted drinking water environment.
(2) Experimental procedure
The experiment sets up two groups of reactors, respectively, control group (no microbial inoculum is added, the blank carrier is used for replacing) and enhancement group (microbial inoculum is added), the two groups all use vetiver grass with similar growth trend and similar root system condition, wherein, based on the total volume of blank luffa filling material added in the field planting basket, the experiment is carried out by adding one tenth proportion of microbial inoculum, the microporous aeration pump is used for aeration in the operation process of the reactor, the dissolved oxygen concentration of the water body is ensured, the water body is simultaneously mixed, the hydraulic retention time is controlled at 24 h, fresh water body is replaced at intervals of 24 h, before and after the water body is replaced, water samples are respectively sampled from upper, middle and lower water intake for 5 ml and mixed to obtain 15 ml water samples which represent the nitrate nitrogen concentration of the whole water environment, the nitrate nitrogen concentration is measured by using a phenol disulfonic acid photometry, and the nitrate nitrogen removal rate is calculated. The overall experimental period was 28 days.
As shown in FIG. 2, the NO was compared between the control group and the enhanced group at the initial stage of the microbial inoculum enhancement (stage 1) 3 - The removal effect of N was similar, NO in the control and boost groups within 1-9 days 3 - The average removal rate of N is 96.82% and 98.03%, and in the early stage of strengthening, the difference is not seen between the control group and the experimental group, and the vetiver is probably fed into the micro-nutrition water body from the non-nutrition water body, the plant accelerates the absorption of nutrition, and the plant is utilized for self growth, so that the strengthening effect of the microbial inoculum cannot be effectively reflected. After day 9 (stage 2), it is possible that the plant spent on the hunger and thirst with nutrients, and the control group on NO 3 - The removal rate of N is reduced from 97.05% on 9 days to 73.73% on 10 days, 23.32% is reduced, the average removal rate on the following 10-28 days is only 72.18%, but the enhanced group has the opposite phenomenon to the control group, the removal efficiency similar to that of the stage 1 is maintained in the stage 2, the average removal rate of the stage 2 is 97.76%, the phenomenon is caused by the enhancement 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 enhancement effect of a system is realized, and the NO of the system is maintained 3 - The removal effect of N ensures that the system is specific to NO 3 - -processing efficiency of N.
While the 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. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the 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 (8)

1. The slow-release carbon source immobilized aerobic denitrification composite microbial agent is characterized by comprising a composite microbial agent and loofah sponge for immobilizing the composite microbial agent, wherein the composite microbial agent comprises Arthrobacter (Arthrobacter sp.) YC-RL1 andAcidovorax sp.the strain YD725 aerobic denitrifying bacteria, wherein (Arthrobacter sp.) YC-RL1 is preserved in China general microbiological culture collection center CGMCC with the preservation number of CGMCC No.10611,Acidovorax sp.the strain YD725 is preserved in China Center for Type Culture Collection (CCTCCM) with the preservation time of 2021, 5 months and 17, and the preservation number of CCTCCM 2021536.
2. The slow-release carbon source immobilized aerobic denitrification compound microbial inoculant according to claim 1, wherein the retinervus Luffae fructus is sodium hydroxide modified retinervus Luffae fructus.
3. The slow-release carbon source immobilized aerobic denitrification composite microbial agent according to claim 1, comprising (archibacter sp.) YC-RL1 and 1:1-2 by weight of the composite microbial agentAcidovorax sp. strain YD725。
4. The preparation method of the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum is characterized by comprising the following steps of:
(1) Providing a bacterial suspension containing an aerobic denitrification composite bacterial agent, wherein the composite bacterial agent comprises (Arthrobacter sp.) YC-RL1 andAcidovorax sp.the strain YD725 aerobic denitrifying bacteria, wherein (Arthrobacter sp.) YC-RL1 strain is preserved in China general microbiological culture collection center CGMCC with the preservation number of CGMCC No.10611,Acidovorax sp.strain YD725 was deposited with China center for type culture Collection for a time of 2021Year 5 and month 17, and the preservation number is CCTCCM 2021536;
(2) Providing sodium hydroxide modified loofah sponge;
(3) Mixing an aqueous solution of polyvinyl alcohol with the bacterial suspension, adding the modified retinervus luffae fructus into the mixed solution, and immobilizing the mixed solution and the modified retinervus luffae fructus under the condition suitable for immobilization to obtain the slow-release carbon source immobilized aerobic denitrification compound bacterial agent.
5. The method for preparing the slow-release carbon source immobilized aerobic denitrification compound microbial inoculum according to claim 4, wherein the mass-volume ratio concentration of the aqueous solution of the polyvinyl alcohol is 0.05-1g/mL.
6. The method for preparing the slow-release carbon source immobilized aerobic denitrification composite microbial inoculum according to claim 5, wherein the condition suitable for immobilization is crosslinking for 12-36h at 0-8 ℃ in the presence of a crosslinking solution, and the crosslinking solution is a saturated boric acid solution of 1-10% calcium chloride.
7. A microbial inoculant-phytoremediation water system, wherein the system comprises an aeration device and a reaction zone, wherein the reaction zone comprises vetiver and a composite inoculant according to any one of claims 1-3.
8. Use of the microbial inoculant-phytoremediation water system of claim 7 in the treatment of pollution of a potable water source.
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