CN113929221B - A microbial complex flora for treating aquaculture tail water - Google Patents

Abstract

The invention provides a microorganism complex flora for treating aquaculture tail water, which comprises bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and micrococcus luteus. The microbial complex flora provided by the invention is used for treating aquaculture tail water. According to the invention, the compound flora is obtained by screening strains and optimizing the number of specific strains, and experimental results show that the obtained compound flora can be used for efficiently treating the culture tail water. In practical application, the flora with the optimal proportion can be fixed on a biological film constructed by brushes, ceramsites and the like, and the effect is good through pilot experiments. The invention fully plays the synergistic effect among bacteria, thereby remarkably improving the treatment effect on the culture water body.

Description

A microbial complex flora for treating aquaculture tail water

technical field

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

Background technique

With the intensive and large-scale development of the aquaculture industry, the problem of tail water pollution has become increasingly serious. Aquaculture tail water contains a large amount of nitrogen and phosphorus pollutants. If it is discharged directly without effective treatment, it is easy to breed bacteria and viruses and cause eutrophication of the water body, causing great damage to the water environment. Therefore, how to efficiently treat the tail water of aquaculture has become an important problem faced by the aquaculture industry and needs to be solved urgently. Among them, the biological treatment method has unique advantages because of its low cost and no secondary pollution.

At present, there are relatively few studies on biological treatment methods, and most of them focus on the treatment of tail water by using a single filter-feeding shellfish, fish or macroalgae. Studies on microbial functional genes have shown that some microorganisms show great advantages in removing nutrient elements, and can absorb elements in water as elements needed for their own growth, which provides theoretical feasibility for them to treat aquaculture tail water.

However, current research is mostly limited to revealing the metabolic potential of a single strain of aquaculture tail water treatment, but the environmental adaptability of a single strain is poor, and the effect in practical applications is often far below expectations. Therefore, it is urgent to find functional strains that specifically remove nitrogen and phosphorus elements, and combine them into a microbial complex flora in a certain ratio, and add them to the tail water treatment system to make them fully exert their treatment efficiency.

Contents of the invention

The purpose of the present invention is to provide a microbial complex flora that can be used to treat aquaculture tail water, thereby effectively improving the treatment efficiency of aquaculture tail water.

The microbial complex flora provided by the present invention comprises Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus licheniformis and Micrococcus luteus;

The quantitative ratio of Micrococcus luteus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium in the microbial complex flora is preferably 2:6:9:5:4.

Application of the microbial composite flora provided by the present invention in the treatment of aquaculture tail water;

The present invention also provides that the provided microbial complex bacteria are also used to prepare biofilms;

The biofilm is a brush or ceramsite attached to and growing the microbial composite bacteria.

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

The present invention obtains a composite flora by screening strains and optimizing the quantity of specific strains, and the experimental results show that the obtained composite flora can efficiently treat aquaculture tail water. In practical applications, the optimal ratio of bacteria can be fixed on the biofilm constructed by brushes, ceramsite, etc., and the effect is verified by pilot experiments. The invention fully exerts the synergistic effect among the bacteria, thereby significantly improving the treatment effect on the aquaculture water body.

Description of drawings

Figure 1: Photos of tail water treated by each experimental group;

Figure 2: The figure of the removal rate of the optimal proportion of bacteria to the pollutants in the tail water of aquaculture;

Figure 3: Pilot test flow chart;

Figure 4: The effect of the system on the treatment of nutrients in the tail water of the culture after adding the complex flora.

Detailed ways

The present invention utilizes microbial composite flora to treat the tail water of aquaculture, and it is found through application effect research that the nutrient salt content in the tail water after the flora treatment is significantly reduced.

The source information of the bacterial strain used in the embodiment of the present invention is as follows:

The Bacillus subtilis (Bacillus subtilis) is preserved in the Beina Biological Microorganisms Collection Bank, and the preservation number is BNCC109047; the preservation address is Xinyang City, Henan Province;

The Bacillus megaterium de Bary is preserved in the Beina Biological Microorganisms Collection, and the preservation number is BNCC336464; the preservation address is Xinyang City, Henan Province;

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is preserved in the Beina Biological Microorganisms Collection Bank, the preservation number is BNCC336388; the preservation address is Xinyang City, Henan Province;

The Bacillus licheniformis (Bacillus licheniformis) is preserved in the Beina Biological Microorganisms Preservation Bank, and the preservation number is BNCC336463; the preservation address is Xinyang City, Henan Province;

The Micrococcus luteus (Micrococcus luteus) is preserved in the Beina Biological Microorganisms Collection Bank, and the preservation number is BNCC102589; the preservation address is Xinyang City, Henan Province;

However, those skilled in the art can also select Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus licheniformis and Micrococcus luteus strains from other sources with similar functions on the basis of the disclosure content of the specification of the present invention.

The present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings.

Example 1: Screening and ratio optimization of bacterial strains in complex flora

Through preliminary experiments, the following 8 strains with good nitrogen and phosphorus removal effects in tail water were determined, namely Bacillus subtilis, Bacillus megaterium de Bary, Bacillus amyloliquefaciens, Bacillus licheniformis Bacillus licheniformis, Micrococcus luteus, Bacillus pumilus, Bacillus flexus, Bacillus cereus. The first five strains were identified by pre-culture experiment screening for further research.

Wherein in the method of the present invention, the indicators of nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total nitrogen, and total phosphorus in the tail water of the breeding water are measured using a SmartChem450 water quality analyzer, and COD is measured using an alkaline potassium permanganate method. Among them, nitrite nitrogen is determined by naphthalene ethylenediamine spectrophotometry, nitrate nitrogen is determined by cadmium column reduction method, phosphate is determined by phosphorus molybdenum blue spectrophotometry method, ammonia nitrogen content is determined by hypobromite oxidation method, and total nitrogen and total phosphorus are determined by persulfuric acid Potassium oxidation method.

1. Single bacteria experiment

Put the pre-cultured bacterial solutions into a centrifuge, centrifuge at 4000rpm for 10 minutes, wash with normal saline three times, and then dilute with sterilized water samples, adjust the OD600 value of the bacterial solution to 0.5, and set the bacterial dosage to five Gradients 1‰, 2‰, 3‰, 4‰, 5‰ were added to 500mL tail water, and the water sample without bacterial solution was used as a blank control. Each sample was set in three parallels, and samples were taken to determine nitrite nitrogen, Nitrate nitrogen, phosphate, ammonia nitrogen, total nitrogen, total phosphorus, and COD indicators are compared to obtain the best removal effect of water body indicators under different dosages.

2. Combination bacteria experiment

The dosage of each single bacteria with the best removal rate for nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total nitrogen, total phosphorus, and COD obtained in the single bacteria experiment is set for the dosage of each single bacteria. According to the level table, an orthogonal experiment with five factors and three levels was designed (Table 2).

Table 1: Orthogonal Design Factors, Level Table

Table 2: Orthogonal experiment analysis table

Put the pre-cultured bacterial solution into a centrifuge, centrifuge at 4000rpm for 10 minutes, wash with normal saline three times, then dilute with sterilized water samples, adjust the OD600 value of the bacterial solution to 0.5, and configure the bacterial suspension according to the level table , put into 1L of cultured tail water in proportion, and take the water sample without adding bacteria solution as the blank control, take the method of putting the Erlenmeyer flask still on the experimental table for the test (Figure 1), and take samples to determine the nitrite nitrogen and nitrate nitrogen. , phosphate, ammonia nitrogen, total nitrogen, total phosphorus, and COD indicators were compared and screened to obtain the best removal effect of different proportions of microbial complex flora on water pollutant indicators (Table 3).

Table 3 The removal rate of each experimental group to the pollutants in the tail water of aquaculture

According to the experimental results, the bacterial group with the best removal effect on nitrite nitrogen is group 11, that is, the ratio of Micrococcus luteus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium is 1:6:7 :5:5. The bacteria group with the best removal effect on nitrate nitrogen was group 2, and the ratio of the bacteria group was 1:6:8:6:4. The bacteria group with the best effect on phosphate removal was group 3, and the ratio of the bacteria group was 1:7:9:7:5. The bacteria group with the best removal effect on ammonia nitrogen was group 13, and the ratio of the bacteria group was 2:5:8:7:3. The bacteria group with the best removal effect on total nitrogen was group 14, and the ratio of the bacteria group was 2:6:9:5:4. The bacterial group with the best removal effect on total phosphorus was group 1, and the bacterial group ratio was 1:5:7:5:3. The best bacterial flora for COD removal were groups 13 and 17, and the flora ratios were 2:5:8:7:3 and 3:6:7:7:3, respectively.

According to the experimental results, considering the three indicators of total nitrogen, total phosphorus, and COD in the tail water of aquaculture as the key factors, among which total nitrogen is the most critical, it is determined that group 14 is the best composite bacterial flora for tail water treatment, that is, Garcinia cambogia The ratio of Micrococcus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium is 2:6:9:5:4. The removal rates of nitrogen, total phosphorus, and COD were 72%, 48%, 32%, 25%, 75%, 6%, and 74% respectively (Figure 2).

Embodiment 2: Preparation of composite flora

First, activate the bacteria, the specific steps are as follows:

①Prepare a test tube containing 5-10mL liquid medium and 2 plates for each type of bacteria, see Table 4 for the preparation method;

② Open the freeze-dried tubes containing 5 kinds of bacteria powder in the safety cabinet, burn the neck with an alcohol lamp, then quickly drop sterile water to break it, and then smash it with tweezers;

③Pipe 0.5mL of liquid culture medium into the freeze-drying tube, dissolve it fully, put it back into the liquid test tube, and mix well;

④Pipe 0.2mL of bacterial suspension into the plate, spread it evenly, and repeat twice to obtain two plates; Then observe the growth of the colony in the liquid medium and the plate, and confirm that the liquid medium is turbid and the shape of the colony in the plate conforms to the morphological characteristics of each bacteria (Table 5), then it can be used.

Table 4: Medium formula table

Table 5: Morphological characteristics of bacteria

Example 3: Utilizing microbial complex flora to biologically treat aquaculture tail water

Put the complex flora into the pilot test to simulate the comprehensive bioremediation system for experiments, and monitor the changes of nutrient content in the tail water during the experiment. Fig. 3 is the flow chart of the pilot test experiment. The system is equipped with 5 processing steps. In addition to the conventional treatment of the sedimentation area, the filter-feeding shellfish area, and the artificial wet area, the optimized flora of the present invention are respectively fixed on the brush area, ceramsite After 10 days of system operation, the tail water treatment effect is shown in Figure 4. It can be seen that the system has a good removal effect on nitrate nitrogen, phosphate, and ammonia nitrogen nutrients after adding complex bacteria. Especially the removal effect of phosphate reached 83.09%, while the system without adding flora was only 38.60%.

The microbial complex flora screened by the invention has good environmental adaptability and good application effect, which can provide new ideas for evaluating and optimizing tail water treatment technology in the future, and contribute to the realization of green development of aquaculture.

Claims (1)

1. the application of a microbial composite flora in the treatment of aquaculture tail water, characterized in that, said composite flora is composed of Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus licheniformis and Micrococcus luteus and the quantitative ratio of Micrococcus luteus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium in the composite flora is 2:6:9:5:4.

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