CN116515710B - Heterotrophic nitrifying bacteria and microbial inoculum, preparation method and application thereof - Google Patents
Heterotrophic nitrifying bacteria and microbial inoculum, preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses heterotrophic nitrifying bacteria bacillus subtilis JHD-Z1, and the biological preservation number is CCTCC NO: m20221913. Also discloses heterotrophic nitrifying bacteria bacillus licheniformis JHD-Z2, the biological preservation number of which is CCTCC NO: m20221914. The invention also discloses a heterotrophic nitrifying bacteria preparation for the aquatic products, which is prepared from bacillus subtilis JHD-Z1 and/or bacillus licheniformis JHD-Z2. The invention also discloses a preparation method of the composite heterotrophic nitrifying bacteria preparation. The bacteria and the microbial inoculum of the invention can not only purify the aquaculture water quality with high efficiency and reduce the ammonia nitrogen and/or nitrite content of the aquaculture water, but also can remarkably improve the growth performance of the cultured animals and provide a good growth environment for the aquaculture animals.
Description
Technical Field
The invention relates to the field of aquatic animal culture and the technical field of microorganism application, in particular to a preparation method and application of a composite heterotrophic nitrifying bacteria preparation for aquatic products.
Background
The increase in demand of aquatic products, one of the important sources of protein acquired by humans, has also prompted the development of intensive farming modes. However, as the cultivation density is continuously increased, the environmental pollution problem is more serious, so it is urgent to find a way capable of maintaining the sustainable development of the aquatic industry. The traditional cultivation mode generally keeps the excellent quality of water by changing water in a large quantity, but the defects of high cost, excessive environmental pollution, low biological safety and the like are also revealed. Ammonia nitrogen generated in the cultivation process is decomposed and absorbed mainly through photosynthesis of algae and nitrification of autotrophic microorganisms, but the algae is easily influenced by factors such as illumination weather, the action process is unstable, the nitrification rate of the autotrophic microorganisms is low, the ammonia nitrogen is rapidly accumulated due to improvement of the feeding rate along with the cultivation process, and the effect of water purification cannot be achieved due to low conversion capability.
The heterotrophic nitrifying bacteria can remove nitrogen in the water body in the modes of absorption, utilization, conversion and the like, so that the water quality can be effectively improved, and meanwhile, the inorganic nitrogen can be directionally, efficiently and reliably utilized. Under the condition of adding a carbon source, the heterotrophic microorganism converts ammonia nitrogen into mycoprotein through heterotrophic transformation, and the water body environment can be quickly and efficiently improved under the proper C/N condition. Meanwhile, the addition of the carbon source can promote the growth of algae, bacteria and protozoa in the water, and the heterotrophic bacteria are taken as main forces to act together, so that ammonia nitrogen in the water is removed. The probiotics are also rich in nutrient elements such as rich proteins, vitamins, minerals and the like, and can be used as an additional nutrient source for cultured animals, so that the feeding amount of the feed and the protein content in the feed are reduced, the cost is saved, and meanwhile, the damage to the water environment can be reduced. Meanwhile, the probiotics have various active substances with the function of promoting growth, so that not only can the nutrition level of the cultured animals be improved, but also the immunity of the animals under the stress condition can be improved. And the metabolites of microorganisms and bacterial cell wall components can be used as immunostimulants to improve the immune function of the cultured animals. The probiotics can also improve the activity of digestive enzymes by colonizing in the intestinal tract of the organism, improve the microbial structure of the intestinal tract, reduce the abundance of potential pathogenic bacteria and ensure the health of the organism.
With the rapid development of aquaculture industry, the variety of corresponding microecologics on the market is increased, but the denitrification performance of each type is different, and the reason is different heterotrophic microorganism types. Therefore, it is important to find a suitable heterotrophic nitrifying bacterium with a high functionality. At present, microecological preparations for aquatic products are generally divided into a single strain preparation and a compound strain preparation, and the compound strain preparation can degrade harmful substances in water more efficiently through symbiotic or cooperative relationship among strains, and has better environmental stress resistance compared with a single strain. Meanwhile, different strains are formed, and different proportions of the strains can influence the denitrification efficiency of the composite bacterial liquid. The proper fermentation condition is used as a prerequisite for the good growth of the strain, and has great influence on the denitrification performance of the composite bacterial liquid. Therefore, it is very important to find a composite heterotrophic nitrifying bacteria with better purification effect on water quality and the optimal denitrification condition thereof.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art and provides two heterotrophic nitrifying bacteria which are more efficient and have stronger water quality purification capability for aquatic products.
Another technical problem to be solved by the present invention is to provide the heterotrophic nitrifying bacteria preparation.
The invention further provides a preparation method and application of the heterotrophic nitrifying bacteria preparation.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the invention provides heterotrophic nitrifying bacteria bacillus subtilis (Bacillus subtilis) JHD-Z1, and the biological preservation number is CCTCC NO: m20221913.
The invention also provides heterotrophic nitrifying bacteria bacillus licheniformis (Bacillus licheniformis) JHD-Z2, and the biological preservation number is CCTCC NO: m20221914.
The invention provides a heterotrophic nitrifying bacteria preparation for aquatic products, which contains bacillus subtilis (Bacillus subtilis) JHD-Z1 and/or bacillus licheniformis (Bacillus licheniformis) JHD-Z2.
The technical problems to be solved by the invention can be realized by the following technical proposal. The heterotrophic nitrifying bacteria preparation for aquatic products is characterized in that the total number of viable bacteria of the bacillus subtilis (Bacillus subtilis) JHD-Z1 in the heterotrophic nitrifying bacteria preparation for aquatic products is x and 1×10 8 ≤x≤1×10 9 The method comprises the steps of carrying out a first treatment on the surface of the The total number of viable bacteria of Bacillus licheniformis (Bacillus licheniformis) JHD-Z2 is y and 1×10 8 ≤y≤1×10 9 。
The technical problems to be solved by the invention can be realized by the following technical proposal. The heterotrophic nitrifying bacteria preparation for the aquatic products is characterized in that when the heterotrophic nitrifying bacteria preparation for the aquatic products contains bacillus subtilis JHD-Z1 and bacillus licheniformis JHD-Z2 at the same time, the weight ratio of the bacillus subtilis JHD-Z1 and the bacillus licheniformis JHD-Z2 is 1:1-3.
The technical problems to be solved by the invention can be realized by the following technical proposal. The heterotrophic nitrifying bacteria preparation for aquatic products is characterized by further comprising an acceptable nutritional dilution carrier, wherein the nutritional dilution carrier is preferably selected from sucrose, naCl and MgSO 4 ·7H 2 O、K 2 HPO 4 One or more of the following.
The invention also provides a preparation method of the heterotrophic nitrifying bacteria preparation for the aquatic products, which is characterized by comprising the following steps of:
(1) The bacillus subtilis JHD-Z1 and the bacillus licheniformis JHD-Z2 are subjected to mutual antagonism test in an oxford cup mode, and the antagonistic strains are not subjected to mixed culture; mixing and culturing the strains in the same proportion for 12h, measuring the concentration of the bacterial liquid by using a turbidimeter, and adjusting the concentration of the bacterial liquid to 4.5X10 7 -4.8×10 7 CFU/mL; inoculating into nitrifying bacteria and nitrosate bacteria liquid culture medium according to the adding amount of 1%, and shake culturing at 30deg.C at 180r/min, pH8 and C/N15 for 96 hr; sampling every 48 hours, and measuring the change of ammonia nitrogen and nitrite concentration in the culture medium;
nitrifying bacteria liquid culture medium: naNO 2 0.0375g，CH 3 COONa 5g，NaCl 1.0g，MgSO 4 ·7H 2 O 1.0g，K 2 HPO 4 1.0g, 1000mL of distilled water;
nitrosation bacteria liquid medium: NH (NH) 4 Cl 4g，CH 3 COONa 5g, vickers salt solution 50mL, distilled water 1000mL, pH 7.5-8.0;
vickers salt solution: k (K) 2 HPO 4 5g，MgSO 4 ·7H 2 O 2.5g，NaCl 2.5g，FeSO 4 ·7H 2 O 0.05g，MnSO 4 ·4H 2 O0.05 g, distilled water 1000mL;
the method for measuring ammonia nitrogen adopts a salicylic acid spectrophotometry, and the method for measuring nitrite nitrogen adopts an N- (1-naphthyl) diethylamine spectrophotometry;
(2) Will 10 7 、10 8 、10 9 CFU/mL is used as different bacterial liquid concentrations, inoculated into nitrifying bacteria and nitrosating bacteria liquid culture media according to the addition amount of 1%, and subjected to shaking culture for 96 hours at the temperature of 30 ℃ at the rotating speed of 180r/min, pH of 8 and C/N15; sampling once in 0, 12, 24, 48 and 96 hours, measuring the change of ammonia nitrogen and nitrite concentration in the culture medium, and determining the optimal bacterial liquid concentration;
(3) Determining the optimal bacterial liquid adding concentration according to the data of the step (2), wherein the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, and the C/N15 is subjected to shake culture for 12 hours, sampling is carried out every 3 hours, the change of ammonia nitrogen and nitrite concentration in a culture medium is determined, and the optimal carbon source is determined;
(4) Setting the C/N as 10, 15, 20 and 25, determining the optimal bacterial liquid concentration and carbon source according to the data of the step (2) and the step (3), carrying out shake culture for 12 hours at the temperature of 30 ℃ at the rotating speed of 180r/min and the pH value of 8, sampling once every 3 hours, measuring the change of the concentration of ammonia nitrogen and nitrite in the culture medium, and determining the optimal C/N;
(5) 1%, 2%, 5% and 10% are used as inoculum size of the bacterial liquid, the optimal bacterial liquid addition concentration, the optimal carbon source and the optimal C/N obtained in the step (2) to the step (4) are inoculated into a liquid culture medium according to different inoculum sizes, the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the shaking culture is carried out for 12 hours, sampling is carried out every 3 hours, the change of the concentration of ammonia nitrogen and nitrite in the culture medium is measured, and the optimal bacterial liquid inoculum size is determined;
(6) Setting the initial pH of a culture medium to 7, 8, 9 and 10 after the adjustment of HCl and NaOH solutions are added into the culture medium, inoculating the optimal bacterial liquid addition concentration, the optimal carbon source, the optimal C/N and the optimal inoculation amount obtained in the step (2) to the step (5) into a liquid culture medium, performing shaking culture at the temperature of 30 ℃ and the rotating speed of 180r/min for 12 hours, sampling once every 3 hours, measuring the change of ammonia nitrogen and nitrite concentration in the culture medium, and determining the optimal initial pH;
(7) Setting the temperature of 25 ℃,30 ℃, 35 ℃ and 40 ℃ as the culture temperature, inoculating the optimal bacterial liquid obtained in the step (2) to the step (6) into a liquid culture medium according to the optimal bacterial liquid addition concentration, the optimal carbon source, the optimal C/N, the optimal inoculum size and the optimal pH, shaking and culturing for 12h, sampling once every 3h, measuring the change of ammonia nitrogen and nitrite concentration in the culture medium, and determining the optimal culture temperature;
(8) Centrifuging and collecting thalli respectively through the final fermentation liquid obtained in the steps (2) to (7) at the speed of 8000rpm for 10min, and respectively weighing the thalli into the thalli according to the weight ratio of the thalli to the protective agent solution: adding a protective agent solution into thalli in a weight ratio of protective agent to 1:5-10, uniformly mixing to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain a composite heterotrophic nitrifying bacteria bacterial agent;
the protectant solution comprises the following components: 25-35g/L of skim milk powder, 15-30g/L of desalted whey powder, 10-20g/L of industrial trehalose, 3-4g/L of vitamin C, 0.04-0.08g/L of lecithin and the balance of distilled water;
the invention also discloses the heterotrophic nitrifying bacteria or the composite heterotrophic nitrifying bacteria preparation for aquatic products or the heterotrophic nitrifying bacteria preparation for aquatic products prepared by the method, which is characterized by reducing the ammonia nitrogen and/or nitrite content of the water for aquaculture and improving the water quality. Meanwhile, the growth performance of the cultured animals can be improved.
The heterotrophic nitrifying bacteria bacillus subtilis (Bacillus subtilis) JHD-Z1 is preserved in China Center for Type Culture Collection (CCTCC) in the year 2022, 12 and 09, and the biological preservation number is CCTCC NO: m20221913. Address: wuhan, university of Wuhan.
The heterotrophic nitrifying bacteria bacillus licheniformis (Bacillus licheniformis) JHD-Z2 is preserved in China Center for Type Culture Collection (CCTCC) with a biological preservation number of CCTCC NO: m20221914. Address: wuhan, university of Wuhan.
Compared with the prior art, the invention has the following beneficial effects:
the 2 heterotrophic nitrifying bacteria obtained by screening are all strains with strong degradation capability, so that the water quality purifying capability is ensured.
The heterotrophic nitrifying bacteria can quickly occupy ecological niches in water after being enriched in a large quantity, and meanwhile, growth of pathogenic microorganisms is restrained by occupying growth space and nutrient substances of the pathogenic microorganisms, so that pathogenic bacteria abundance in a system is reduced. Both the bacterial metabolites and the cell wall components can be used as immunostimulants to enhance the immune function of farmed animals. The probiotics can also be planted in the intestinal canal of the organism, so that the digestive enzyme activity is improved, the intestinal microbial structure is improved, the abundance of potential pathogenic bacteria is reduced, the health of the organism is ensured, and the growth performance of the cultured animals is improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below.
Example 1, a composite heterotrophic nitrifying bacteria preparation for aquatic products, comprises the following specific operation steps:
s1, carrying out mutual antagonism test on bacillus subtilis JHD-Z1 and bacillus licheniformis JHD-Z2 by adopting an oxford cup mode, wherein the antagonistic strains are not subjected to mixed culture. Respectively using purified strains as indicator bacteria, coating 200 μl of the indicator bacteria on 2216E solid culture medium, placing oxford cups around the plate, adding 100 μl of other bacterial liquid into oxford cups, and the bacterial liquid concentrations are 10 7 CFU/mL, cultured at 30℃for 24h. As a result, no transparent ring was observed near the oxford cup, and mixed fermentation was possible.
S2, after antagonism test, 2 strains are mixed and cultured for 12 hours according to the same proportion, and the concentration of the bacterial liquid is measured by a turbidimeter and is regulated to 4.5-4.8x10 7 CFU/mL. Inoculating into nitrifying bacteria and nitrosating bacteria liquid culture medium according to the adding amount of 1%, and shake culturing at 30deg.C at 180r/min, pH8 and C/N15 for 96 hr. Sampling is carried out every 48 hours, and the change of ammonia nitrogen and nitrite concentration in the culture medium is measured. Nitrifying bacteria liquid culture medium: naNO 2 0.0375g(NO 2 - -N 10mg/L)，CH 3 COONa 5g，NaCl 1.0g，MgSO 4 ·7H 2 O 1.0g，K 2 HPO 4 1.0g, 1000mL of distilled water. Nitrosation bacteria liquid medium: NH (NH) 4 Cl 4g(NH 4 + -N 50mg/L)，CH 3 COONa 5g, vickers salt solution 50mL, distilled water 1000mL, pH 7.5-8.0. Vickers salt solution: k (K) 2 HPO 4 5g，MgSO 4 ·7H 2 O 2.5g，NaCl 2.5g，FeSO 4 ·7H 2 O 0.05g，MnSO 4 ·4H 2 O0.05 g, distilled water 1000mL. The determination method of ammonia nitrogen adopts salicylic acid spectrophotometry, and the determination method of nitrite nitrogen adopts N- (1-naphthyl) diethylamine spectrophotometry.
TABLE 1 degradation rates of heterotrophic nitrifying bacteria preparations on ammonia nitrogen and nitrite
The results are shown in Table 1, and the degradation capacity of the composite heterotrophic nitrifying bacteria preparation on ammonia nitrogen is obviously higher than that of each single heterotrophic nitrifying bacteria preparation at 48 and 96 hours. The degradation capacity of each group to nitrite is not obviously different, and the degradation rate reaches 100% in 96 hours. Thus, the heterotrophic nitrifying bacteria have synergistic effect, and the denitrification performance is better after mixed culture.
The denitrification performance of the composite heterotrophic nitrifying bacteria under 6 conditions of different bacteria liquid concentrations, different carbon sources, different C/N, different inoculum sizes, different pH values and different temperatures is studied.
S3, respectively 10 7 、10 8 、10 9 CFU/mL is used as different bacterial liquid concentrations, inoculated into nitrifying bacteria and nitrosating bacteria liquid culture media according to the addition amount of 1%, and subjected to shaking culture for 96 hours at the temperature of 30 ℃ at the rotating speed of 180r/min, pH of 8 and C/N15. Sampling is carried out once in 0, 12, 24, 48 and 96 hours, and the change of ammonia nitrogen and nitrite concentration in the culture medium is measured.
TABLE 2 influence of different bacterial liquid concentrations on degradation rates of Ammonia nitrogen and nitrite
The results are shown in Table 2, at 0-12h,10 7 The degradation rate of the treatment group to ammonia nitrogen and nitrite is lowest, and only about 51.3% and 28%. Next, 10 8 In the treatment group, both degradation rates reach about 80%. 10 9 The degradation capacity of the treatment group is strongest, and the degradation rate of the treatment group reaches 100% in 12 hours. The degradation rate of ammonia nitrogen and nitrite in each group reaches 100% in 24 hours. And at 96h,10 9 The degradation capacity of ammonia nitrogen and nitrite in the treatment group is all thatDescending. Comprehensively judge that the concentration of the bacterial liquid is 10 8 The denitrification performance is optimal.
S4, adding the bacterial liquid with the concentration of 10 8 And (3) when CFU/mL is carried out, sodium acetate, sucrose, sodium succinate and sodium citrate are selected as unique carbon sources, the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the C/N15 is subjected to shake culture for 12 hours, sampling is carried out every 3 hours, and the change of the concentration of ammonia nitrogen and nitrite in the culture medium is measured.
TABLE 3 influence of different carbon sources on degradation rates of Ammonia nitrogen and nitrite
As shown in Table 3, the sucrose group has the best degradation capacity on ammonia nitrogen and nitrite, the degradation efficiency reaches about 84.93% and 97.67% respectively at 12h, and the degradation rate of sodium succinate group on ammonia nitrogen is almost stable at 6-12h and is at a lower level of about 20%. Thus, the denitrification performance is optimal when the carbon source is sucrose.
S5, adding the bacterial liquid with the concentration of 10 8 When CFU/mL and the carbon source is sucrose, the addition amount of the carbon source is changed to enable the C/N to be 10, 15, 20 and 25 respectively, the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the shaking culture is carried out for 12 hours, sampling is carried out every 3 hours, and the change of the concentration of ammonia nitrogen and nitrite in the culture medium is measured.
TABLE 4 influence of different carbon to nitrogen ratios on degradation rates of ammonia nitrogen and nitrite
As shown in Table 4, the degradation rates were highest at 20C/N and found to be 82.3% and 91.8%, respectively. And when the C/N is 10, the degradation capacity of ammonia nitrogen and nitrite is lower than that of other groups. Although the degradation capacity of nitrite at 12h is only slightly lower than that of group 20 at 15C/N, the degradation capacity of nitrite is significantly different from that of group 20 at C/N. Therefore, the denitrification performance is best when the C/N is 20.
S6, adding the bacterial liquid with the concentration of 10 8 When CFU/mL and carbon source are sucrose and C/N is 20, 1%, 2%, 5% and 10% are selected as inoculum size of the bacterial liquid, the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the culture is carried out for 12 hours by shaking, sampling is carried out every 3 hours, and the change of ammonia nitrogen and nitrite concentration in the culture medium is measured.
TABLE 5 influence of different inoculum sizes on degradation rate of ammonia nitrogen and nitrite
The results are shown in Table 5, where the inoculum size was 2%, the ammonia nitrogen degradation capacity was superior to that of the other groups at each time point, whereas the nitrite degradation rate was shown to be lower at 0-9h, significantly lower than in the 5% and 10% treatment groups, but the degradation rate was rapidly increased at 9-12h, and was not significantly different from that in the 12h and 10% treatment groups. Thus, the denitrification performance is optimal when the inoculum size is 2%.
S7, adding the bacterial liquid with the concentration of 10 8 When CFU/mL, the carbon source is sucrose, the C/N is 20, and the inoculation amount is 2%, the initial pH is adjusted to 7, 8, 9 and 10 by adding HCl and NaOH solution into the culture medium, the temperature is 30 ℃, the rotating speed is 180r/min, the culture is carried out for 12 hours by shaking, sampling is carried out every 3 hours, and the change of the concentration of ammonia nitrogen and nitrite in the culture medium is measured.
TABLE 6 influence of different pH on degradation rate of Ammonia nitrogen and nitrite
As shown in Table 6, the degradation ability was optimal at pH 9, and the degradation ability to ammonia nitrogen and nitrite was 100% or so at pH 9, so that the denitrification performance was optimal at pH 9.
S8, adding the bacterial liquid with the concentration of 10 8 CFU/mL, sucrose as carbon source, 20C/N, inoculum size of 2%, pH 9Setting four culture temperatures of 25 ℃,30 ℃, 35 ℃ and 40 ℃, carrying out shaking culture for 12 hours at 180r/min, sampling every 3 hours, and measuring the change of ammonia nitrogen and nitrite concentration in a culture medium.
TABLE 7 influence of different temperatures on degradation rate of ammonia nitrogen and nitrite
As a result, as shown in Table 7, the degradation rate reached 100% at 9 hours at 30℃and was far higher than that at 25 ℃. The degradation rate of ammonia nitrogen at the temperature of 35 and 40 ℃ reaches 100% in 12 hours, but the degradation capacity of nitrite is far lower than that of the group at the temperature of 30 ℃. The degradation capacity of ammonia nitrogen and nitrite is the lowest at 25 ℃. Therefore, 30 ℃ is selected as the optimal temperature for fermentation of the bacterial liquid;
to obtain that when the bacterial liquid adding concentration is 10 8 CFU/mL, sucrose as carbon source, 20C/N, 2% inoculum size, 9 pH and 30 ℃ for the heterotrophic nitrifying bacteria.
S9, fermenting the composite heterotrophic nitrifying bacteria for 12 hours under the optimal fermentation condition, centrifuging at 8000rpm for 10min to collect thalli, adding a protective agent solution into the thalli according to the weight ratio of the thalli to the protective agent solution of the weight ratio of the thalli to the protective agent of the weight ratio of 1:5-10 respectively, uniformly mixing to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain the composite heterotrophic nitrifying bacteria bacterial agent, wherein the protective agent solution comprises the following components: 25-35g/L of skim milk powder, 15-30g/L of desalted whey powder, 10-20g/L of industrial trehalose, 3-4g/L of vitamin C, 0.04-0.08g/L of lecithin and the balance of distilled water.
Example 2 application effect evaluation experiment of composite heterotrophic nitrifying bacteria preparation for aquatic products, the steps are as follows:
s1, japanese lobster is obtained from Ganjiao aquatic products development Limited company of Jiangsu province, litsea japonica, and has an average body mass of (2.55+ -0.18) g and an average body length of (6.4+ -0.09) cm. The crude protein content of the compound feed for the Penaeus japonicus is 49.01%, the crude fat content is 7.24%, and the C/N in the feed is about 11;
s2, after the breeding objects are retrieved, placing the breeding objects into a 1000L breeding barrel for temporary breeding for 7 days, wherein the temperature is 30+/-0.5 ℃, the salinity is 28+/-0.3, the pH is 9.1+/-0.1, and the dissolved oxygen is 7+/-0.2 mg/L. During temporary rearing, pellet feed is fed twice a day (8:00 and 19:00), the feeding amount is 3% of the weight of the shrimps, and the soil pick-up treatment is carried out 1h after feeding. Water was changed 1/3 of the day during the temporary rearing period. After temporary culture, 300 Penaeus japonicus with similar specifications and good vitality are evenly distributed into 10 culture boxes (60L, 50cm multiplied by 40cm multiplied by 30 cm) for experiment. The experiment set controls and the experiment set, each set was set with 5 parallels. The culture conditions of the control group are consistent with the temporary culture conditions, and the experimental group adopts a zero water changing culture mode and only supplements evaporation loss water. The composite heterotrophic nitrifying bacteria obtained by adopting the experimental combination are fermented for 12 hours according to the optimal culture condition and then are put into a water body, so that the initial concentration of the bacteria in the water body is kept between 4.0 and 4.5 multiplied by 10 8 CFU/mL. The experimental group added sucrose daily as an organic carbon source to maintain a C/N (mass concentration) of 20:1 in the water. The experimental period is 32d;
s3, measuring water quality indexes every 4d in the experimental period, wherein the influence of the culture water quality is shown in a table 8. The ammonia nitrogen and nitrate content of the experimental group is obviously higher than that of the control group (P < 0.05) at the 4d, and then is obviously reduced, the ammonia nitrogen, nitrite and nitrate content is obviously reduced from the 8d and is lower than that of the control group (P < 0.05), and the ammonia nitrogen, nitrite and nitrate content is kept at a lower level as the experiment progresses. The ammonia nitrogen and nitrate contents of the control group show fluctuation type growth along with the time, and the nitrite contents always show rising trend. The pH of the control group and the pH of the experimental group are in a descending trend along with the cultivation, and the descending trend of the experimental group is more obvious;
TABLE 8 influence of composite heterotrophic nitrifying bacteria on aquaculture water quality
Note that: the same row of data shoulder marks show that the difference is obvious (P is less than 0.05), the same letters of the shoulder marks or no shoulder marks show that the difference is not obvious (P is more than 0.05)
S4, counting the growth performance of the Penaeus japonicus after the experiment is finished, and the results are shown in Table 9. The survival rate of the Penaeus japonicus in the experimental group reaches 79% and is obviously higher than that in the control group (P is less than 0.05). Meanwhile, the weight gain rate and the specific growth rate of the experimental group are respectively improved by 58.9 percent and 50.7 percent (P is less than 0.05) compared with the control group. The control group and the experimental group have no significant difference in fertilizer fullness (P > 0.05);
TABLE 9 Effect of Compound heterotrophic nitrifying bacteria on growth performance of Penaeus japonicus
Note that: the same row of data shoulder marks show that the difference is obvious (P is less than 0.05), the same letters of the shoulder marks or no shoulder marks show that the difference is not obvious (P is more than 0.05)
S5, randomly selecting 9 shrimps from each treatment group after the experiment is finished, collecting the tissues of hepatopancreas, muscle, stomach and intestinal canal, preparing a mixed sample from the same tissues of every 3 shrimps, accurately weighing, placing the mixed sample into a 1.5mL centrifuge tube, transferring the mixed sample into liquid nitrogen for quick freezing, and then transferring the mixed sample to a refrigerator at the temperature of-80 ℃ for later use. According to the mass of each tissue sample, adding 0.9% physiological saline according to the mass-volume ratio of 1:9 to prepare 10% tissue homogenate, centrifuging at 4 ℃ for 10min at 2500r/min, and taking supernatant to perform enzyme activity measurement. Measuring nonspecific immune enzyme activities in hepatopancreas and muscle tissue, including superoxide dismutase (WST-1 method), catalase, alkaline phosphatase, and acid phosphatase; digestive enzyme activities in stomach and intestinal tissues including trypsin, pepsin, lipase, alpha-amylase were measured. The enzyme activity detection kit is purchased from Nanjing's institute of biological engineering.
TABLE 10 Effect of Compound heterotrophic nitrifying bacteria on non-specific immunoenzymatic Activity of Penaeus japonicus
Note that: the same column data shoulder marks have obvious difference (P is less than 0.05), the same letters of the shoulder marks or no shoulder marks have insignificant difference (P is more than 0.05)
Table 11 Effect of Compound heterotrophic nitrifying bacteria on digestive enzyme Activity of Penaeus japonicus
Note that: the same column data shoulder marks have obvious difference (P is less than 0.05), the same letters of the shoulder marks or no shoulder marks have insignificant difference (P is more than 0.05)
The results are shown in tables 10-11, and the activities of acid phosphatase, alkaline phosphatase, catalase and superoxide dismutase in the muscle and hepatopancreatic tissues of the experimental group are higher than those of the control group, and the significant difference (P < 0.05) exists; the activities of pepsin, trypsin, alpha-amylase and lipase in the experimental group are obviously higher than those in the control group (P is less than 0.05) in the stomach and intestinal tissues, which shows that the compound heterotrophic nitrifying bacteria can improve the immune and digestion functions of the Penaeus japonicus;
in conclusion, the composite heterotrophic nitrifying bacteria preparation obtained by the method has better water quality purifying capacity compared with the original finished microecological preparation. The method is characterized in that the method is obtained by combining various indexes, and the addition concentration of the bacterial liquid is 10 8 The CFU/mL, the carbon source is sucrose, the C/N is 20, the inoculation amount is 2%, the pH is 9, and the temperature is 30 ℃, so that the composite heterotrophic nitrifying bacteria obtained by the method can be used for more rapidly and efficiently denitrifying. Meanwhile, by combining with a culture experiment, the composite heterotrophic nitrifying bacteria preparation can promote the growth of the Japanese prawns, improve the growth performance of the Japanese prawns and improve the digestion and immune functions.
Claims (8)
1. A heterotrophic nitrifying bacteria preparation for aquatic products is characterized in that the bacteria preparation contains the following componentsBacillus grassBacillus subtilis) JHD-Z1 and bacillus licheniformis%Bacillus licheniformis) JHD-Z2; the bacillus subtilis is [ ]Bacillus subtilis) The biological preservation number of JHD-Z1 is CCTCC NO: m20221913; the bacillus licheniformis is [ ]Bacillus licheniformis) The biological preservation number of JHD-Z2 is CCTCC NO: m20221914.
2. The heterotrophic nitrifying bacteria preparation for aquatic products according to claim 1, wherein the bacillus subtilis isBacillus subtilis) The total number of viable bacteria of JHD-Z1 is x and 1×10 8 ≤x≤1×10 9 The method comprises the steps of carrying out a first treatment on the surface of the Bacillus licheniformis [ ]Bacillus licheniformis) The total number of live bacteria of JHD-Z2 is y and 1×10 8 ≤y≤1×10 9 。
3. The heterotrophic nitrifying bacteria preparation for aquatic products according to claim 1, wherein the weight ratio of the bacillus subtilis JHD-Z1 to the bacillus licheniformis JHD-Z2 is 1:1-3.
4. The heterotrophic nitrifying bacteria preparation for aquatic products according to claim 1, further comprising an acceptable nutritional dilution carrier.
5. The heterotrophic nitrifying bacteria preparation for aquatic products according to claim 4, wherein said nutrient-diluting carrier is selected from the group consisting of sucrose, naCl, mgSO 4 ·7H 2 O、K 2 HPO 4 One or more of the following.
6. A method for preparing the heterotrophic nitrifying bacteria preparation for aquatic products according to claim 1, comprising the following steps:
the mutual antagonism test between strains of bacillus subtilis JHD-Z1 and bacillus licheniformis JHD-Z2 is carried out by adopting an oxford cup mode, and the strains producing antagonism are notPerforming mixed culture; the strain is mixed and cultured in the same proportion for 12h, and the concentration of the bacterial liquid is measured by a turbidimeter and adjusted to 4.5X10 7 -4.8×10 7 CFU/mL; inoculating into nitrifying bacteria and nitrosating bacteria liquid culture medium according to the addition amount of 1%, and shake culturing at 30deg.C, rotation speed of 180r/min, pH8 and C/N15 for 96h; sampling every 48 to h, and measuring the change of ammonia nitrogen and nitrite concentration in the culture medium;
nitrifying bacteria liquid culture medium: naNO 2 0.0375 g，CH 3 COONa 5 g，NaCl 1.0 g，MgSO 4 ·7H 2 O 1.0 g，K 2 HPO 4 1.0g, distilled water 1000mL;
nitrosation bacteria liquid medium: NH (NH) 4 Cl 4g，CH 3 COONa 5g, vickers salt solution 50mL, distilled water 1000mL, pH 7.5-8.0;
vickers salt solution: k (K) 2 HPO 4 5 g，MgSO 4 ·7H 2 O 2.5 g，NaCl 2.5 g，FeSO 4 ·7H 2 O 0.05 g，MnSO 4 ·4H 2 O0.05 g, distilled water 1000mL;
the method for measuring ammonia nitrogen adopts a salicylic acid spectrophotometry, and the method for measuring nitrite nitrogen adopts an N- (1-naphthyl) diethylamine spectrophotometry;
will 10 7 、10 8 、10 9 CFU/mL is used as different bacterial liquid concentrations, inoculated into nitrifying bacteria and nitrosating bacteria liquid culture media according to the addition amount of 1%, and subjected to shaking culture at the temperature of 30 ℃ at the rotating speed of 180r/min, pH of 8 and C/N15 of 96h; sampling once at 0, 12, 24, 48 and 96h, measuring the change of ammonia nitrogen and nitrite concentration in the culture medium, and determining the optimal bacterial liquid concentration;
determining the optimal bacterial liquid adding concentration according to the data of the step (2), wherein the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the C/N15 shake culture is 12h, sampling is carried out every 3h, the change of ammonia nitrogen and nitrite concentration in the culture medium is determined, and the optimal carbon source is determined;
(4) Setting the concentration of the optimal bacterial liquid and the carbon source as 10, 15, 20 and 25 according to the data of the step (2) and the step (3), carrying out shake culture at the temperature of 30 ℃ at the rotating speed of 180r/min and the pH value of 8 for 12h, sampling once every 3h, measuring the concentration change of ammonia nitrogen and nitrite in the culture medium, and determining the optimal C/N;
(5) 1%, 2%, 5% and 10% are used as inoculum size of the bacterial liquid, the optimal bacterial liquid addition concentration, the optimal carbon source and the optimal C/N obtained in the step (2) to the step (4) are inoculated into a liquid culture medium according to different inoculum sizes, the temperature is 30 ℃, the rotating speed is 180r/min, the pH is 8, the shaking culture is 12h, sampling is carried out every 3h, the change of ammonia nitrogen and nitrite concentration in the culture medium is measured, and the optimal bacterial liquid inoculum size is determined;
(6) The initial pH of the culture medium is set to 7, 8, 9 and 10 after being regulated by adding HCl and NaOH solutions into the culture medium, the optimal bacterial liquid addition concentration, the optimal carbon source, the optimal C/N and the optimal inoculation amount obtained in the step (2) to the step (5) are inoculated into a liquid culture medium, the temperature is 30 ℃, the rotating speed is 180r/min, the shaking culture is carried out for 12h, sampling is carried out every 3h, the change of ammonia nitrogen and nitrite concentration in the culture medium is measured, and the optimal initial pH is determined;
(7) Setting the temperature of 25 ℃,30 ℃, 35 ℃ and 40 ℃ as the culture temperature, inoculating the optimal bacterial liquid obtained in the step (2) to the step (6) into a liquid culture medium according to the optimal bacterial liquid adding concentration, the optimal carbon source, the optimal C/N, the optimal inoculum size and the optimal pH, carrying out shaking culture for 12h at 180r/min, sampling once every 3h, measuring the concentration change of ammonia nitrogen and nitrite in the culture medium, and determining the optimal culture temperature;
(8) Centrifuging and collecting thalli respectively through 8000rpm and 10min of the final fermentation liquid obtained in the steps (2) to (7), adding a protective agent solution into thalli respectively according to the weight ratio of thalli to the protective agent solution of the thalli weight of (5-10) which is the weight ratio of the protective agent to 1, uniformly mixing to obtain a bacterial suspension, and freeze-drying the bacterial suspension to obtain the composite heterotrophic nitrifying bacteria microbial inoculum;
the protectant solution comprises the following components: 25-35g/L of skim milk powder, 15-30g/L of desalted whey powder, 10-20g/L of industrial trehalose, 3-4g/L of vitamin C, 0.04-0.08g/L of lecithin and the balance of distilled water.
7. Use of a complex heterotrophic nitrifying bacteria preparation for aquatic products according to any one of claims 1-5 or a heterotrophic nitrifying bacteria preparation for aquatic products according to the method of claim 6, characterized in that said use is to reduce the ammonia nitrogen and/or nitrite content of aquaculture water and improve the water quality.
8. Use of a composite heterotrophic nitrifying bacteria preparation for aquatic products according to any one of claims 1-5 or a heterotrophic nitrifying bacteria preparation for aquatic products according to the method of claim 6 for improving the growth performance of farmed animals.
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| CN109650556A (en) * | 2019-01-23 | 2019-04-19 | 武汉丰甜生物科技有限公司 | The composite bacillus microbial inoculum and its application of ammonia nitrogen and nitrite in degradation water body |
| CN110372105A (en) * | 2019-07-11 | 2019-10-25 | 中国科学院烟台海岸带研究所 | A kind of complex micro organism fungicide and preparation method thereof for improveing aquaculture system |
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| CN1632105A (en) * | 2004-12-02 | 2005-06-29 | 沈锦玉 | Bacillus subtilis preparation |
| CN103937712A (en) * | 2014-04-04 | 2014-07-23 | 华南理工大学 | Bacillus licheniformis and application thereof |
| CN109650556A (en) * | 2019-01-23 | 2019-04-19 | 武汉丰甜生物科技有限公司 | The composite bacillus microbial inoculum and its application of ammonia nitrogen and nitrite in degradation water body |
| CN110372105A (en) * | 2019-07-11 | 2019-10-25 | 中国科学院烟台海岸带研究所 | A kind of complex micro organism fungicide and preparation method thereof for improveing aquaculture system |
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