CN113929221B

CN113929221B - Microorganism composite flora for treating aquaculture tail water

Info

Publication number: CN113929221B
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: 2023-07-14
Anticipated expiration: 2041-11-22
Also published as: CN113929221A

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

Microorganism 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 microorganism composite flora for treating aquaculture tail water.

Background

Along with the intensive and large-scale development of the aquaculture industry, the tail water pollution problem brought by the aquaculture industry is increasingly serious. The cultivation tail water contains a large amount of nitrogen and phosphorus pollutants, and if the cultivation tail water is not effectively treated and directly discharged, bacteria, viruses and water eutrophication are easy to breed, and the water area environment is greatly damaged. Therefore, how to treat the culture tail water efficiently becomes an important problem facing the culture industry, and needs to be solved. Among them, the biological treatment method has unique advantages due to its low cost and no secondary pollution.

The current research on biological treatment methods is relatively few and is focused on treating tail water with a single filter-feeding shellfish, fish or macroalgae. The research of functional genes of microorganisms shows that partial microorganisms show great advantage of removing nutrient elements, and can absorb elements in water body as elements required by self growth, so that theoretical feasibility is provided for the treatment of aquaculture tail water.

However, the current research is limited in revealing the metabolic potential of a single strain on the treatment of the culture tail water, but the environmental adaptation of the single strain is poor, and the effect in practical application is often far from being expected. Therefore, functional strains for removing nitrogen and phosphorus elements in a targeted manner are needed to be found out, and are combined into a microorganism composite flora according to a certain proportion, and the microorganism composite flora is added into a tail water treatment system so as to fully exert the treatment efficiency.

Disclosure of Invention

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

The microbial composite 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 microorganism composite flora is preferably 2:6:9:5:4.

the application of the microorganism complex flora provided by the invention in the treatment of aquaculture tail water;

the invention also provides a preparation method of the microbial composite bacteria;

the biological film is a brush or ceramsite which is adhered and grown with the microorganism composite bacteria.

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

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.

Drawings

Fig. 1: treating tail water photo graphs of each experimental group;

fig. 2: a removal rate diagram of the optimal proportion of flora on the pollutants in the tail water of the cultivation;

fig. 3: pilot plant experiment flow chart;

fig. 4: and after the complex flora is added, the system is used for treating nutrient salt in the tail water of the cultivation.

Detailed Description

According to the invention, the microbial composite flora is utilized to treat the culture tail water, and the application effect research shows that the nutrient salt content in the tail water body treated by the microbial composite flora is obviously reduced.

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

the bacillus subtilis (Bacillus subtilis) is preserved in a North Nabiological microorganism strain preservation library, and the preservation number is BNCC109047; the preservation address is Xinyang city of Henan province;

the bacillus megatherium (Bacillus megaterium de Bary) is preserved in a North Nabiological microorganism strain preservation library, and the preservation number is BNCC336464; the preservation address is Xinyang city of Henan province;

the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is preserved in a North Nana microorganism strain preservation library, and the preservation number is BNCC336388; the preservation address is Xinyang city of Henan province;

the bacillus licheniformis (Bacillus licheniformis) is preserved in a North Nami microorganism strain preservation library, and the preservation number is BNCC336463; the preservation address is Xinyang city of Henan province;

the micrococcus luteus (Micrococcus luteus) is preserved in a North Nabiological microorganism strain preservation library, and the preservation number is BNCC102589; the preservation address is Xinyang city of Henan province;

however, other sources of bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis, and micrococcus luteus strains with similar functions may be selected by those skilled in the art based on the disclosure of the present invention.

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

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

The following 8 strains with better effect of removing nitrogen and phosphorus in tail water are respectively determined by early experiments, namely Bacillus subtilis (Bacillus subtilis), bacillus megaterium (Bacillus megaterium de Bary), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus licheniformis (Bacillus licheniformis), micrococcus luteus (Micrococcus luteus), bacillus pumilus (Bacillus pumilus), bacillus curvatus (Bacillus flexus) and Bacillus cereus (Bacillus cereus). The first 5 bacteria were screened for further study by pre-culture experiments.

The indexes of nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total nitrogen and total phosphorus in the culture tail water in the method are measured by using a SmartChem450 water quality analyzer, and COD is measured by using an alkaline potassium permanganate method. Wherein the nitrosamine is spectrophotometrically prepared by naphthalene ethylenediamine, the nitrosamine is reduced by cadmium column, the phosphate is spectrophotometrically prepared by phosphomolybdenum blue, the ammonia nitrogen content is measured by hypobromite oxidation, and the total nitrogen and total phosphorus are oxidized by potassium persulfate.

1. Single fungus experiment

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

2. Combined bacterium experiment

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

Table 1: orthogonal design factor, level gauge

Table 2: orthogonal experimental analysis table

The bacterial liquid which is cultivated in advance is put into a centrifuge for centrifugation at 4000rpm for 10min, then the bacterial liquid is washed for three times by using normal saline, then the bacterial liquid is diluted by using a sterilized water sample, the OD600 value of the bacterial liquid is regulated to be 0.5, bacterial suspension is prepared according to a level meter, the bacterial liquid is put into 1L of cultivation tail water according to a proportion, a water sample without bacterial liquid is taken as a blank control, a mode of standing a conical flask on a laboratory table for test is adopted (figure 1), indexes of nitrite nitrogen, nitrate nitrogen, phosphate, ammonia nitrogen, total phosphorus and COD are sampled and measured and compared, and the optimal removal effect of microbial compound flora with different proportions on the indexes of water pollutants is obtained by screening (table 3).

Table 3 removal rates of the various Experimental groups on the contamination of the culture tail water

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

according to experimental results, 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 key, so that the group 14 is determined to be the optimal complex flora for treating the tail water, namely micrococcus luteus, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and bacillus megaterium, and the proportion of the micrococcus luteus to the bacillus subtilis to the bacillus amyloliquefaciens is 2:6:9:5:4, the removal rates of the complex bacterial group 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 microbial flora

Firstly, activating bacteria, which comprises the following specific steps:

(1) 1 test tube containing 5-10mL liquid culture medium and 2 plates are prepared for each strain, and the preparation method is shown in Table 4;

(2) the freeze-drying pipes filled with 5 bacterial powders are respectively opened in the safety cabinet, the top part is burnt by an alcohol lamp, then sterile water is quickly dropped to break the top part, and then the top part is broken by tweezers;

(3) sucking 0.5mL of liquid culture medium, pumping into a freeze-drying tube, fully dissolving, pumping back into the liquid test tube, and uniformly mixing;

(4) sucking 0.2mL of bacterial suspension, pouring the bacterial suspension into a flat plate, uniformly coating the bacterial suspension, and repeating the steps twice to obtain two flat plates; (5) the liquid test tube and plate were all placed under the following conditions: after the bacterial strain grows out, the growth conditions of bacterial colonies in the liquid culture medium and the flat plate are observed, and the turbidity of the liquid culture medium and the conformation of the bacterial colonies in the flat plate are confirmed to be consistent with the morphological characteristics of each bacterial strain (table 5), the bacterial strain can be used.

Table 4: medium formulation table

Table 5: form characteristic table of bacteria

Example 3: treatment of aquaculture tail water using microorganism composite flora organisms

And (3) putting the composite flora into a pilot scale experiment simulation comprehensive bioremediation system for experiment, and monitoring the content change of nutritive salt in tail water in the experiment process. FIG. 3 is a flow chart of a pilot experiment, the system is provided with 5 processing steps, besides a conventional processing sedimentation area, a filter feeding shellfish area and an artificial wet area, the preferable post-bacterial colony of the invention is respectively fixed in a brush area and a ceramsite area, 100L of aquaculture tail water is processed by using the preferable post-bacterial colony, the tail water processing effect is shown in FIG. 4 after the system is operated for 10 days, the system has better removal effect on nitrate nitrogen, phosphate and ammonia nitrogen nutritive salt after adding the compound bacterial colony, especially the removal effect of 83.09% is achieved by the phosphate, and the system without adding the bacterial colony is only 38.60%.

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

Claims

1. The application of the microorganism composite flora in the treatment of aquaculture tail water is characterized in that the composite flora consists of bacillus subtilis, bacillus megaterium, bacillus amyloliquefaciens, bacillus licheniformis and micrococcus luteus; and 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.