CN113929221A

CN113929221A - Microbial composite flora for treating aquaculture tail water

Info

Publication number: CN113929221A
Application number: CN202111389787.1A
Authority: CN
Inventors: 陈文杰; 郑忠明; 杨文�; 魏颖祯
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-01-14
Anticipated expiration: 2041-11-22
Also published as: CN113929221B

Abstract

The invention provides a microbial composite flora for treating aquaculture tail water, which comprises bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and micrococcus luteus. The microbial composite flora provided by the invention is used for treating aquaculture tail water. The invention obtains the compound flora by screening strains and optimizing the number of specific strains, and the experimental result shows that the obtained compound flora can efficiently treat the culture tail water. In practical application, the flora with the optimal proportion can be fixed on a biological membrane constructed by brushes, ceramsite and the like, and the experimental results prove that the effect is good. The invention fully exerts the synergistic effect among the bacteria, thereby obviously improving the treatment effect on the aquaculture water body.

Description

Microbial composite flora for treating aquaculture tail water

Technical Field

The invention belongs to the technical field of environmental treatment microorganisms, and particularly relates to a microbial composite flora for treating aquaculture tail water.

Background

With the intensive and large-scale development of aquaculture industry, the problem of tail water pollution caused by the aquaculture industry is increasingly severe. The culture tail water contains a large amount of nitrogen and phosphorus pollutants, and if the culture tail water is directly discharged without effective treatment, bacteria and viruses are easy to breed, water eutrophication is easy to occur, and the environment of the water area is greatly damaged. Therefore, how to treat the culture tail water with high efficiency becomes an important difficult problem in the culture industry, and solution is urgently needed. Among them, the biological treatment method has unique advantages due to its low cost and no secondary pollution.

Currently, biological treatment methods are relatively poorly studied and focus on treating tailwater with a single filter-feeding shellfish, fish, or macroalgae. Research on microbial functional genes shows that part of microbes show great advantage of removing nutrient elements, and can absorb elements in a water body into elements required by growth of the microbes, so that theoretical feasibility is provided for treating aquaculture tail water.

However, the current research is limited to reveal the metabolic potential of a single strain on the treatment of the culture tail water, but the environmental adaptability of the single strain is poor, and the effect in practical application is often far lower than expected. Therefore, it is urgently needed to find out functional strains for removing nitrogen and phosphorus elements, combine the functional strains into a microbial composite flora according to a certain proportion, and add the microbial composite flora into a tail water treatment system so as to fully exert the treatment efficiency.

Disclosure of Invention

The invention aims to provide a microbial composite flora for treating aquaculture tail water, so that the treatment efficiency of the aquaculture tail water is effectively improved.

The microbial compound flora provided by the invention comprises bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and micrococcus luteus;

the number ratio of micrococcus luteus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus megaterium in the microbial composite flora is preferably 2: 6: 9: 5: 4.

the application of the microbial composite flora provided by the invention in treating aquaculture tail water;

the invention also provides the microbial composite bacteria which are also used for preparing a biological membrane;

the biological film is a brush or ceramsite attached with the microorganism composite bacteria.

The invention also provides application of the microbial composite flora in preparation of an aquaculture tail water treatment system.

The invention obtains the compound flora by screening strains and optimizing the number of specific strains, and the experimental result shows that the obtained compound flora can efficiently treat the culture tail water. In practical application, the flora with the optimal proportion can be fixed on a biological membrane constructed by brushes, ceramsite and the like, and the experimental results prove that the effect is good. The invention fully exerts the synergistic effect among the bacteria, thereby obviously improving the treatment effect on the aquaculture water body.

Drawings

FIG. 1: photograph of treated tail water of each experimental group;

FIG. 2: a removal rate graph of the optimal proportion of flora to the culture tail water pollutants;

FIG. 3: a pilot test experimental flow chart;

FIG. 4: the effect of the system after adding the complex flora on the nutrient salt in the culture tail water is shown.

Detailed Description

According to the invention, the culture tail water is treated by utilizing the microbial composite flora, and the research on the application effect shows that the content of nutritive salt in the tail water body after the flora treatment is obviously reduced.

The source information of the strains used in the examples of the present invention is as follows:

the Bacillus subtilis is preserved in a northern Nami biological microorganism strain preservation library with the preservation number of BNCC 109047; the preservation address is Xinyang city of Henan province;

the Bacillus megaterium (Bacillus megaterium de Bary) is preserved in a northern Nami biological microorganism strain preservation library, and the preservation number is BNCC 336464; the preservation address is Xinyang city of Henan province;

the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is preserved in a Beinanna biological microorganism strain preservation library with the preservation number of BNCC 336388; the preservation address is Xinyang city of Henan province;

the Bacillus licheniformis (Bacillus licheniformis) is preserved in a Beina biological microorganism strain preservation library, and the preservation number is BNCC 336463; the preservation address is Xinyang city of Henan province;

the Micrococcus luteus (Micrococcus luteus) is preserved in a Beina biological microorganism strain preservation library with the preservation number of BNCC 102589; the preservation address is Xinyang city of Henan province;

however, those skilled in the art can select other strains of Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus licheniformis and Micrococcus luteus with similar functions based on the disclosure of the present invention.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: screening and proportion optimization of strains in complex flora

Through earlier experiments, the following 8 strains with good effects of removing nitrogen and phosphorus in tail water are respectively Bacillus subtilis (Bacillus subtilis), Bacillus megaterium de Bary, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus licheniformis (Bacillus licheniformis), Micrococcus luteus (Micrococcus luteus), Bacillus pumilus (Bacillus pumilus), Bacillus flexus (Bacillus flexus) and Bacillus cereus (Bacillus cereus). The first 5 strains were screened by a preculture experiment and further investigated.

In the method, indexes of nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total nitrogen and total phosphorus in the culture tail water are measured by using a SmartChem450 water quality analyzer, and COD is measured by using an alkaline potassium permanganate method. Wherein the nitrite nitrogen is measured by naphthyl ethylenediamine spectrophotometry, the nitrate nitrogen is reduced by cadmium column, the phosphate is measured by phosphomolybdium blue spectrophotometry, the ammonia nitrogen content is measured by hypobromite oxidation, and the total nitrogen and the total phosphorus are measured by potassium persulfate oxidation.

1. Single bacterium experiment

Putting each pre-cultured bacterial liquid into a centrifuge, centrifuging for 10min at 4000rpm, washing for three times by using physiological saline, diluting by using a sterilized water sample, adjusting the OD600 value of the bacterial liquid to 0.5, setting five gradients of 1 per mill, 2 per mill, 3 per mill, 4 per mill and 5 per mill for the bacterial feeding amount, feeding the bacterial liquid into 500mL tail water, taking the water sample without the bacterial liquid as a blank control, setting three samples in parallel, sampling and measuring nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total phosphorus and COD indexes, and comparing to obtain the optimal removal effect of the water body indexes under different feeding amounts.

2. Combined bacteria experiment

The addition amount of each single bacterium obtained in the single bacterium experiment with the optimal removal rate on nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total phosphorus and COD is set to be three different inoculation amount per mill levels (table 1) for the addition amount of each single bacterium, and a five-factor three-level orthogonal test (table 2) is designed according to the level table.

Table 1: orthogonal design factor, horizon table

Table 2: orthogonal experimental analysis table

Putting the pre-cultured bacterial liquid into a centrifuge, centrifuging at 4000rpm for 10min, washing with physiological saline for three times, diluting with a sterilized water sample, adjusting the OD600 value of the bacterial liquid to 0.5, preparing bacterial suspension according to a horizontal surface, putting into 1L of culture tail water in proportion, taking the water sample without the bacterial liquid as a blank control, statically placing a conical flask on an experiment table for testing (figure 1), sampling to measure nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total phosphorus and COD indexes, comparing, and screening to obtain the optimal removal effect of the microbial composite flora with different proportions on the water body pollutant indexes (Table 3).

TABLE 3 removal rate of tail water pollutants from cultivation in each experimental group

According to the experimental results, the flora with the best effect of removing nitrite nitrogen is group 11, namely micrococcus luteus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus megaterium, the proportion of which is 1: 6: 7: 5: 5. the flora with the best effect of removing nitrate and nitrogen is group 2, the flora ratio is 1: 6: 8: 6: 4. the group of colonies with the best phosphate removal effect was group 3, the ratio of colonies was 1: 7: 9: 7: 5. the flora with the best ammonia nitrogen removal effect is group 13, and the flora ratio is 2: 5: 8: 7: 3. the group of colonies with the best effect on total nitrogen removal was group 14, the ratio of colonies was 2: 6: 9: 5: 4. the flora with the best effect of removing total phosphorus is group 1, the flora ratio is 1: 5: 7: 5: 3. the groups 13 and 17 were the best groups for COD removal, with a ratio of 2: 5: 8: 7: 3 and 3: 6: 7: 7: 3.

according to the experimental result, three indexes of total nitrogen, total phosphorus and COD in the culture tail water are considered as key factors, wherein the total nitrogen is the most key, so that the optimal compound flora for treating the tail water in the group 14 is determined, namely, the proportion of micrococcus luteus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus megaterium is 2: 6: 9: 5: 4, the removal rates of the composite flora on nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total phosphorus and COD are respectively 72%, 48%, 32%, 25%, 75%, 6% and 74% (figure 2).

Example 2: preparation of Complex flora

Firstly, activating the bacteria, and specifically comprising the following steps:

1 test tube containing 5-10mL of liquid culture medium and 2 flat plates are respectively prepared for various bacteria, and the preparation method is shown in a table 4;

opening freeze-drying tubes filled with 5 kinds of fungus powder in the safety cabinet, burning the top by an alcohol lamp, then quickly dripping sterile water to break the top, and then breaking the top by tweezers;

pumping 0.5mL of liquid culture medium into a freeze-drying tube, pumping the liquid culture medium back into the liquid test tube after the liquid culture medium is fully dissolved, and uniformly mixing;

fourthly, sucking 0.2mL of bacterial suspension, injecting the bacterial suspension into a flat plate, uniformly coating the bacterial suspension, and repeating the steps twice to obtain two flat plates; placing the liquid test tube and the flat plate under the following conditions: and (3) aerobic culture at 30 ℃, standing for 24h, observing the growth conditions of bacterial colonies in the liquid culture medium and the flat plate after the bacterial strains grow out, and determining that the liquid culture medium is turbid and the morphology of the bacterial colonies in the flat plate conforms to the morphological characteristics of each bacterium (Table 5) before use.

Table 4: culture medium formula table

Table 5: form characteristic table of bacteria

Example 3: biological treatment of aquaculture tail water by using microbial composite flora

And (3) putting the composite flora into a pilot-scale experiment simulation comprehensive bioremediation system for experiment, and monitoring the content change of the nutritive salt in tail water in the experiment process. Fig. 3 is a flow chart of a pilot plant experiment, 5 treatment steps are arranged in the system, except a conventional treatment settling zone, a filter feeding shellfish zone and an artificial wet area, the optimized floras of the invention are respectively fixed in a hairbrush zone and a ceramsite zone, 100L aquaculture tail water is treated by the floras, and after the system runs for 10 days, the treatment effect of the tail water is shown in fig. 4, so that the system has a good removal effect on nitrate nitrogen, phosphate and ammonia nitrogen nutritive salt after the compound floras is added, particularly the removal effect of the phosphate reaches 83.09%, and the system without the floras is only 38.60%.

The screened microorganism composite flora has good environmental adaptability and good application effect, can provide a new idea for evaluating and optimizing tail water treatment technology in the future, and contributes to the green development of aquaculture.

Claims

1. The microbial composite flora is characterized in that the composite flora comprises bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and micrococcus luteus.

2. The microbial composite flora of claim 1, wherein the number ratio of micrococcus luteus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus megaterium in the composite flora is 2: 6: 9: 5: 4.

3. use of a complex population of microorganisms according to claim 1 or 2 for the treatment of aquaculture tail water.

4. Use of a complex population of microorganisms according to claim 1 or 2 for the preparation of a biofilm.

5. A biofilm which is a brush or a ceramsite on which the complex microbial flora according to claim 1 or 2 is attached and grown.

6. Use of the complex microbial consortia of claim 1 or 2 in the preparation of an aquaculture tail water treatment system.