WO2021077662A1 - 一种聚氨酯生物膜及凡纳滨对虾高密度标苗和养殖的方法 - Google Patents

一种聚氨酯生物膜及凡纳滨对虾高密度标苗和养殖的方法 Download PDF

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WO2021077662A1
WO2021077662A1 PCT/CN2020/079551 CN2020079551W WO2021077662A1 WO 2021077662 A1 WO2021077662 A1 WO 2021077662A1 CN 2020079551 W CN2020079551 W CN 2020079551W WO 2021077662 A1 WO2021077662 A1 WO 2021077662A1
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polyurethane
biofilm
litopenaeus vannamei
seedling
nitrite
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PCT/CN2020/079551
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English (en)
French (fr)
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宋志文
刘佳
刘超
徐爱玲
乔凤禄
陆继哲
唐敬超
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青岛理工大学
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Priority to KR1020227017191A priority Critical patent/KR20220093136A/ko
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/50—Culture of aquatic animals of shellfish
    • A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • the invention relates to the technical field of Litopenaeus vannamei seedlings and breeding technology, in particular to a polyurethane biofilm and a high-density Litopenaeus vannamei seedlings and a breeding method.
  • the standard seedlings of Litopenaeus vannamei are also called "standard rough", which refers to purchasing 3-10 day-old larvae from the seedling farm to strengthen and desalinate them to reach a body length of more than 1 cm, and adjust the salinity of the pond. The process of reaching the range required by the farmers. Target seedlings are of great significance for improving the survival rate of shrimp seedlings, shortening the grow-out time, grow-out management and scientific feeding.
  • water purification is the primary problem to be solved, and the key to the problem is the removal of ammonia and nitrite in the water body. Due to the large breeding density of Litopenaeus vannamei in the breeding stage, it is easy to cause excessive ammonia and nitrite in the water body, resulting in slow growth of the shrimp larvae, susceptible to disease, and low survival rate.
  • the methods to improve the water quality of the seedling process mainly include a large amount of water exchange, biofloc technology (BFT) and recirculating aquaculture systems (RAS) and so on. Frequent water changes not only waste water resources and pollute the environment, but also cause stress to shrimp larvae.
  • BFT biofloc technology
  • RAS recirculating aquaculture systems
  • the recirculating aquaculture system model is considered to be an environmentally friendly aquaculture model, but its investment, operating costs and technical requirements are high, making it difficult to promote in a large area.
  • Biological flocculation technology adjusts the C/N ratio by adding organic carbon sources, increases the number of heterotrophic bacteria in the water, uses microorganisms to assimilate inorganic nitrogen, and the flocs are ingested twice by the prawns to achieve regulation of water quality, promote nutrient circulation, and reduce feed coefficients.
  • the biofilm method mainly adds biofilm carriers to the system to allow nitrifying microorganisms to adhere and grow. It can not only effectively remove nitrogen compounds, especially highly toxic ammonia and nitrite, and improve the survival and growth rate of shrimps, but also The formed biofilm can be used as an additional food source for shrimp, providing essential nutrients such as unsaturated fatty acids, amino acids and vitamins.
  • Nitrifying microorganisms mainly include ammonia oxidizing microorganisms (ammonia oxidizing bacteria AOB and ammonia oxidizing archaea AOA) and nitrite oxidizing bacteria (NOB).
  • AOB and AOA convert ammonia into nitrite, and NOB further converts nitrite into Low toxicity nitrate.
  • AOB, AOA and NOB are autotrophic microorganisms, their generation time, slow reproduction speed, and sensitivity to changes in environmental factors make them at a disadvantage in the competition with heterotrophic bacteria. If the biofilm filler is directly added to the breeding (standard seedling) pond, the water quality will change regularly during the filming process.
  • heterotrophic bacteria there are a certain number of heterotrophic bacteria, ammonia oxidizing microorganisms and nitrite oxidizing bacteria in the breeding (standard seedling) pond.
  • the heterotrophic bacteria gradually become active, and while absorbing and transforming organic matter, they release ammonia and ammonia. Oxidizing microorganisms then convert ammonia into nitrite. Because the reproduction rate of these two types of microorganisms is very different, the generation time of ammonia oxidizing microorganisms is 24 to 36 hours, while the heterotrophic bacteria is only about 20 minutes, so the initial number of them is too wide. As the heterotrophic bacteria multiply, the ammonia concentration continues to increase.
  • the ammonia content exceeds the absorption and consumption range of the ammonia oxidizing microorganisms, the ammonia quickly accumulates in the water body, and gradually rises to a peak. After that, as the ammonia oxidizing microorganisms continued to proliferate, the ammonia oxidation effect gradually increased, the ammonia concentration began to gradually decrease, and then the nitrite concentration began to gradually increase, and the nitrite oxidizing bacteria began to play a role. Compared with ammonia oxidizing bacteria, nitrite oxidizing bacteria have a longer generation time, so the conversion of nitrite is slower, causing nitrite to gradually accumulate and peak.
  • the seedling time is generally 20 to 30 days, and the density of shrimp seedlings is high, depending on the direct addition of fillers and the natural filming process, the nitrite will continue to increase in the later stage of the seedling, causing the shrimp seedlings to become sick or die.
  • the purpose of the present invention is to overcome the difficulties in water quality control, low survival rate and slow growth of shrimp larvae in the existing Litopenaeus vannamei marking methods, and to fully consider the living habits of Litopenaeus vannamei, the space of the breeding pond and the nitrification. function on the basis of characteristics of the microorganisms, and the use of the biofilm to provide a polyurethane which was vannamei standard high density and seedling culture method having no external organic carbon source, good quality stability, preventing the invasion of pathogenic bacteria, reduce shrimp
  • the morbidity rate can increase or decrease the number of stuffing bags according to the concentration of pollutants, and it is convenient to use.
  • a polyurethane biomembrane which can be used for high-density breeding and breeding of Litopenaeus vannamei.
  • the polyurethane biomembrane uses polyurethane foam as a biomembrane carrier, and ammonia oxidizing microorganisms and nitrite oxidizing bacteria are attached to form a biomembrane.
  • the polyurethane biomembrane uses polyurethane foam as the biomembrane carrier, and through intermittent aeration and energy supply to regulate the proliferation process of ammonia oxidizing microorganisms and nitrite oxidizing bacteria, the pre-formation has a high
  • the nitrifying active biofilm has a nitrite oxidation rate>1.0mg/g ⁇ h, and the ratio of the nitrite oxidation rate to the ammonia oxidation rate is 4-8.
  • ammonia oxidizing microorganisms include ammonia oxidizing bacteria AOB and ammonia oxidizing archaea AOA.
  • AOB and AOA can convert ammonia into nitrite, and nitrite oxidizing bacteria (NOB) further reduce nitrite Converted into low-toxicity nitrate.
  • NOB nitrite oxidizing bacteria
  • Both ammonia oxidizing functional microorganisms and nitrite oxidizing bacteria can choose the existing reported bacterial species, as long as they can achieve their functions.
  • the nitrifying bacteria preparation prepared by Qingdao Haiyisheng Environmental Technology Co., Ltd. is selected. Of course, under the content of the present invention, similar strains or bacterial agents can also be selected.
  • polyurethane foam is a type of high molecular polymer formed by polymerization and foaming of isocyanate and hydroxyl compound.
  • the polyurethane biofilm is prepared by placing the polyurethane foam in artificial seawater, adding nitrifying bacteria preparations (containing ammonia oxidizing function microorganisms and nitrite oxidizing bacteria) to regulate its proliferation process, A biofilm is formed, and the nitrite oxidation rate is >1.0 mg/g ⁇ h, and the ratio of the nitrite oxidation rate to the ammonia oxidation rate is 4-8.
  • the polyurethane foam is added at 20%-40% (V/V) compared to artificial seawater. Under this ratio, the adsorption characteristics of the foam porous material can be more conveniently utilized, which is beneficial to improve the adhesion of the biofilm. Quantity and adhesion efficiency.
  • the method of regulating the proliferation process after adding nitrifying bacteria is: adding sodium nitrite at a concentration of 50-300 mg/L of nitrous nitrogen, and measuring the concentration of nitrous nitrogen every 12 hours.
  • concentration is lower than 0.5mg/L
  • add sodium nitrite again add 4-6 times in total
  • ammonium chloride at a concentration of 20-50 mg/L, and measure the ammonia nitrogen concentration every 12 hours, when the ammonia nitrogen concentration is less than 0.1mg
  • add ammonium chloride again add 1 to 2 times in total to complete the culturing process of polyurethane biofilm.
  • the composition of ammonia oxidizing microorganisms and nitrite oxidizing bacteria in the polyurethane biofilm can be well regulated, and the biofilm can quickly and effectively achieve the nitrite oxidation rate>1.0mg/g ⁇ h, and The ratio of nitrite oxidation rate to ammonia oxidation rate is 4-8.
  • nitrifying bacteria after adding nitrifying bacteria to the preparation method of polyurethane biofilm, it also includes adding trace element liquid and yeast extract to meet the requirements of nitrifying bacteria (containing ammonia oxidizing function microorganisms and nitrite oxidizing bacteria).
  • the composition of the trace element solution is: zinc sulfate 0.287mg, copper sulfate 7.5mg, manganese chloride 14.85mg, ferric chloride 5.38g, sodium molybdate 6.8mg, ethylenediamine Disodium tetraacetate 2.4mg, cobalt chloride hexahydrate 12mg, nickel sulfate 2.4mg, distilled water 1000ml.
  • the salinity of artificial seawater is in the range of 3 ⁇ to 45 ⁇
  • the addition amount of the nitrifying bacteria preparation is 1% to 3% (v/v)
  • the amount of the trace element liquid is 0.1% (v/v)
  • the addition amount of yeast extract is 0.01% (w/v).
  • the artificial seawater salinity ranges from 15 ⁇ to 30 ⁇ . In the embodiment of the present invention, it is preferable to use seawater to configure artificial seawater.
  • the method for preparing the polyurethane biofilm adopts intermittent aeration to form the biofilm.
  • the polyurethane completes the biofilm forming the fastest time (22 days), and the final AOR and NOR values (ammonia oxidation rate (AOR) and nitrite)
  • AOR ammonia oxidation rate
  • NOR oxidation rate
  • the specific operation process in one embodiment is: stop aeration every 2 to 4 hours for 15 to 30 minutes, during which the temperature and dissolved oxygen (DO) are respectively controlled at 28 to 30°C and 4 to 6 mg/L, and the time lasts for 20 ⁇ 25 days.
  • the present invention also discloses a high-density seedling method of Litopenaeus vannamei based on polyurethane biofilm.
  • the method includes: preparing the above polyurethane biofilm and Litopenaeus vannamei standard seedlings; The ammonia and nitrite produced during the seedling process are converted in time to reduce the accumulation of ammonia and nitrite, and achieve high density standard coarseness of Litopenaeus vannamei.
  • the polyurethane biofilm ie, film-hanging polyurethane foam
  • the pore size of the mesh bag should be smaller than the particle size of the polyurethane foam carrier.
  • the dosage of the polyurethane biofilm is 0.5% to 2.5% (V/V) of the standard seedling water.
  • the material of the mesh bag can be a material with high corrosion resistance such as polyester.
  • pre-filmed polyurethane foam is filled in a polyester mesh bag, the size of the mesh bag is 20-60 liters, the mesh size is 2-5mm, and the mesh bag has a zipper opening to facilitate the filling and taking out of the filler.
  • the net bag is placed in the breeding pond according to the polyurethane biofilm/artificial seawater at 0.5 to 2.5% (V/V).
  • the net bag is placed in the standard seedling pond at the same time Add a gas stone to the bag to supplement the dissolved oxygen to improve the efficiency of nitrification.
  • the temperature is controlled at 28-30°C during the seedling period, the dissolved oxygen is> 6.0 mg/L, the ammonia nitrogen is ⁇ 0.1 mg/L, and the nitrous nitrogen is ⁇ 0.5 mg/L.
  • P5 ⁇ P10 seedlings of Litopenaeus vannamei are placed at a density of 10,000 to 30,000 per cubic meter of water for management of the standard seedling process; The process takes 15 to 30 days, and the Litopenaeus vannamei can be desalinated to below 3 ⁇ salinity.
  • the seedling emergence rate can reach more than 80%, and the size is 1.0-2.5 cm.
  • the seedlings obtained are all vigorous and lively, with full muscles, full intestines, and the whole body is clean and free of attachments. It is sensitive to external stimuli, and has head water swimming and wall sticking behaviors. .
  • P5, P8, P10, etc. are ratings based on the color of shrimp larvae, breeding area, water source environment, etc. The larger the number, the higher the quality.
  • the management of the standard seedling process can be adopted as follows: at the initial stage of desalination (1 to 3 days), feeding Artemia 3 times a day, feeding time is 9:00, 15:00, 21:00, and the amount of feeding is in accordance with 2g per 10,000 tails (95% hatching rate), 3 days later, except for worms, feed 100,000 shrimps with 200-500g per day per day, 5 times per day; after another 3 days Change to feeding shrimp feed only. Keep away from the stuffing bag as much as possible during feeding process. It will be desalinated from the 3rd day after seedling feeding, and the daily salinity will not change more than 1 ⁇ 3 ⁇ .
  • Litopenaeus vannamei is a process of strengthening and desalination cultivation of 3-10 day old larvae.
  • the management of the specific seedling process can also adopt some changes, such as investment Feed time, fade gradient, etc. are not limited to the embodiment of the present invention.
  • the daily inspection indicators include: water temperature, salinity, pH, dissolved oxygen, ammonia nitrogen, nitros nitrogen, etc., so as to facilitate timely management and control of standard seedlings.
  • the polyurethane biofilm can be reused during the seedling process.
  • the polyurethane biofilm film-hanging polyurethane foam filler
  • the polyurethane biofilm is taken out of the mesh bag every 20 to 30 days when the seedlings are marked, and rinsed with water of the same salinity to remove the attached suspension. Then put the stuffing in the bag and put it back into the standard seedling pond for use.
  • the target seedling process is implemented in a breeding tank (bucket).
  • the target seedling tank is equipped with temperature control and aeration devices.
  • the standard seedling pond is enclosed by canvas, two exhaust stones are installed in the pond, and the pond is filled with artificial seawater; the polyurethane biofilm is placed in a polyester mesh bag, and the mesh bag is placed in the middle of the standard seedling pond , And keep it immersed, add air stone to the mesh bag.
  • the size of the standard seedling pond is 0.2-60 cubic meters, such as 2 cubic meters, 10 cubic meters, 30 cubic meters, and so on.
  • the size of the target seedling pond is 10-20 cubic meters, which can more effectively increase the survival rate of shrimp seedlings and reduce the stress.
  • the present invention also discloses a method for cultivating Litopenaeus vannamei based on a polyurethane biofilm.
  • the method prepares the above polyurethane biofilm, and uses the polyurethane biofilm in Litopenaeus vannamei cultivation ponds to remove ammonia nitrogen and nitroso groups. Nitrogen step.
  • the aquaculture pond is added with seawater to make the salinity 0.5 ⁇ -30 ⁇ , and the perforated aeration tube is used for oxygenation in the aquaculture pond, and the dissolved oxygen is controlled at 7.0-8.5 mg/L; the water temperature is controlled at 28 ⁇ 1°C, the polyurethane biofilm (film-hanging polyurethane foam) is filled in the stuffing bag and immersed in the water.
  • the sewage will be discharged every 7-10 days, and the water loss due to evaporation and sewage will be supplemented at the same time.
  • the main detection indicators include: temperature, pH, salinity, dissolved oxygen, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, etc.
  • the ammonia nitrogen and nitros nitrogen of the water body are always at a low level during the breeding process, the ammonia nitrogen is less than 0.1 mg/L, and the nitrous nitrogen is less than 0.5 mg/L.
  • the shrimp survival rate is 81.2% , The bait coefficient is 1.38 respectively.
  • the present invention utilizes the characteristics of attachment growth of ammonia oxidizing microorganisms and nitrite oxidizing bacteria, adopts an adsorption method to fix ammonia oxidizing microorganisms and nitrite oxidizing bacteria, and passes intermittently Aeration and energy material supply regulate the proliferation process of ammonia oxidizing microorganisms and nitrite oxidizing bacteria, so that a certain number and proportion of ammonia oxidizing microorganisms and nitrite oxidizing bacteria grow on the polyurethane to form a stable biofilm, and the polyurethane is determined The size and ratio of the ammonia oxidation rate of the biofilm to the nitrite oxidation rate.
  • Figure 1 Surface morphology changes before and after the polyurethane foam film is mounted and after use (a cube with a side length of 1-2cm); (A) before film mounting; (B) after film mounting; (C) after 10 days of use, (D) 20 after use Diva.
  • Figure 3 is a structural diagram of the seedling pond according to Embodiment 2 of the present invention. 1 beam, 2 water surface, 3 stuffing bags, 4 air stones, 5 valves, and 6 drainage pipes.
  • Fig. 4 shows the changes in the concentration of ammonia nitrogen and nitros nitrogen during the process of the seedlings of the present invention related to Example 2.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.
  • Polyurethane foam film can be achieved through the following steps:
  • AOR or NOR K ⁇ V/W, where K is the slope of the scatter diagram of the concentration of ammonia nitrogen or nitros nitrogen (mg/L), V is the volume of the solution, and W is the dry weight of 4 urethane foam monomers (g) ,
  • K is the slope of the scatter diagram of the concentration of ammonia nitrogen or nitros nitrogen (mg/L)
  • V is the volume of the solution
  • W is the dry weight of 4 urethane foam monomers (g)
  • the final determination of AOR is 0.250mg/g ⁇ h
  • NOR is 1.475mg/g ⁇ h
  • the ratio of NOR to AOR is 5.9.
  • Fluorescence in situ hybridization was used to analyze the distribution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in polyurethane biofilms.
  • AOB used NS0190 probe with sequence 5'-CGATCCCTGCTTTTCTCC-3'. The 5'-end of the needle is labeled with FAM;
  • NOB uses the NIT3 probe with the sequence 5'-5CCTGTGCTCCATGCTCCG-3', the 5'-end of the probe is labeled with HEX, and the competitive probe CNIT3 sequence is 5'-CCTGTGCTCCAGGCTCCG-3'.
  • the test results are shown in Table 2.
  • the abundance of AOB in the polyurethane biofilm is 24.8%, and the abundance of NOB is 72.7% (other proportions due to the fast proliferation of ammonia oxidizing bacteria, there will be a peak of nitrite during the seedling process, resulting in shrimp seedlings. Produce poison). It shows that the method of the invention can well regulate the composition of ammonia oxidizing microorganisms and nitrite oxidizing bacteria in the polyurethane biofilm.
  • polyurethane foam monomers are randomly selected from each group and added to 300ml artificial seawater, 0.1% (v/v) trace element dissolved and 0.01% (w/v) )
  • a 500 ml Erlenmeyer flask of yeast extract add ammonium chloride to make the initial concentration of ammonia nitrogen 5mg/L, then shake flask culture at 150 rpm and 28°C, measure ammonia nitrogen and nitrite nitrogen every 4 hours Concentration, to determine the peak concentration of nitrous nitrogen during the experiment.
  • the experimental results are shown in Table 3.
  • the polyurethane biofilm active filler was sealed and stored in a plastic sealed bag, and the changes in ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) were measured every 1 month. The results are shown in Figure 2, which can be seen from Figure 2. After 1 year, its ammonia oxidation rate (AOR) and nitrite oxidation rate (NOR) can still maintain high activity, which is equivalent to 48% and 55% of the initial value, respectively.
  • the seedling pond is made of canvas material, with an effective volume of 2.0m 3 (length: 2.0m, width: 1.0m, height: 1.2m).
  • a total of 16 air stones are installed in two rows in the pool.
  • the pool was filled with 0.6m high artificial seawater with a salt content of 18 ⁇ .
  • the concentration of ammonia nitrogen in the aquaculture water was always lower than 0.1 mg/L, and the concentration of nitrous nitrogen was lower than 0.2 mg/L in the first 12 days, and then increased, but always lower than 0.5 mg/L; nitrate nitrogen was gradually The rising trend increased from 2.1 mg/L to 8.3 mg/L, and the pH dropped from 7.83 to 7.35.
  • Example 2 150,000 P5 shrimp larvae were put into two standard seedling ponds respectively, and the feeding method of bait was the same as that in Example 2.
  • the desalination started on the 3rd day after the seedlings were introduced.
  • the desalination method of the target seedling pond I was the same as that of Example 2.
  • the water quality change during the entire target seedling process was basically the same as that of Example 2, and the ammonia nitrogen was always below 0.1 mg/L. Nitrous nitrogen is below 0.4 mg/L.
  • the water quality of the standard seedling pond II was relatively stable in the first 5 days, and then the nitrous nitrogen began to rise and rose to 0.5 mg/L on the 8th day. After that, the water had to be changed every day to ensure that the nitrous nitrogen was below 0.5 mg/L. , By the 15th day, the daily water exchange rate gradually increased to 60% to 80%, and the shrimp stress phenomenon was more serious.
  • the seedling pond I was cultivated to start dividing seedlings on the 25th day.
  • the average body length of shrimp larvae is 2.1cm, the average weight is 0.18g, and the survival rate is 86.4%. Due to the large amount of water exchange in the later stage, the shrimps were under severe stress in the seedling pond II.
  • the seedling process ended after only 16 days of culture.
  • the average body length of the shrimp larvae was 1.1cm, the average weight was 0.12 grams, and the survival rate was 55.7%.
  • This example further verified the use effect of polyurethane biofilm in Litopenaeus vannamei culture.
  • a cement pond with a water volume of 20 cubic meters was selected for the experiment of Litopenaeus vannamei culture.
  • the size of the culture pond was 5.0 meters ⁇ 4.0 meters ⁇ 1.2 meters, and the water depth was 1.0 meters.
  • the perforated aeration tube is used for oxygenation in the aquaculture pond, and the dissolved oxygen is 7.0-8.5 mg/L.
  • the water temperature is controlled at 28 ⁇ 1°C.
  • the main detection indicators include: temperature, pH, salinity, dissolved oxygen, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, etc.
  • the ammonia nitrogen and nitros nitrogen of the water body are always at a low level, the ammonia nitrogen is less than 0.1 mg/l, and the nitros nitrogen is less than 0.5 mg/l.
  • the shrimp survival rate is 81.2%, and the feed coefficient is 1.38 respectively.

Abstract

本发明公开了一种聚氨酯生物膜及凡纳滨对虾高密度标苗和养殖的方法，本发明以聚氨酯泡沫作为生物膜载体；标苗过程在配备控温、曝气装置以及悬挂填料袋横梁的养殖池或养殖桶实现；利用海水素配制所需盐度人工海水；用孔径2～5毫米的网袋将挂膜聚氨酯填料悬挂在凡纳滨对虾标苗池中；按照密度1～3万尾/立方米水体投放凡纳滨对虾P5～P10苗，进行标苗过程管理；整个标苗过程为15～30天，可将凡纳滨对虾淡化至3‰盐度以下；标苗结束后，将填料袋冲洗去除悬浮物质后可重复使用。该方法利用海水素配制所需盐度人工海水，用预先挂膜的聚氨酯泡沫去除水体中氨和亚硝酸盐等有毒物质，无需添加有机碳源，实现凡纳滨对虾高密度标粗。

Description

一种聚氨酯生物膜及凡纳滨对虾高密度标苗和养殖的方法 技术领域
本发明涉及凡纳滨对虾标苗和养殖技术领域，尤其涉及的是一种聚氨酯生物膜及凡纳滨对虾高密度标苗和养殖的方法。
背景技术
中国是世界上最大的水产养殖国，养殖年产量居世界各国之首，其中对虾养殖面积在400万亩以上。凡纳滨对虾(Litopenaeus vannamei)，也称南美白对虾，因其含肉率高、肉质鲜美，广受消费者喜爱，为世界三大经济虾种之一，具有生长快、养殖周期短、环境适应范围广、饵料要求低、抗逆性强、品质高、可冰鲜运输、加工性能好等优点，可以采取纯淡水、半咸水、海水多种养殖模式，产量占世界养殖虾总产量的70％。自1988年引入中国，经历了30年发展，已成为国内水产养殖的支柱性产业，年产量最高峰时达160多万吨，占全国养殖对虾总产量90％，占世界养殖产量50％，年产值逾500亿元。
凡纳滨对虾标苗也称“标粗”，是指将种苗场购进3～10日龄仔虾进行促壮淡化培育，使其达到体长1厘米以上，并将出池盐度调整到养殖户要求范围的过程。标苗对于提高虾苗成活率，缩短养成时间，养成管理和科学投饵有着重要意义。在标苗过程中，水质净化是首要解决的问题，而问题的关键是水体中氨和亚硝酸盐的去除。由于凡纳滨对虾标苗阶段养殖密度大，容易造成水体中氨和亚硝酸盐超标，导致虾苗生长缓慢，易受病害影响，成活率低。
目前改善标苗过程水质的方法主要有大量换水、生物絮团技术(Biofloc technology，BFT)和循环水养殖系统模式(Recirculating aquaculture systems，RAS)等。频繁换水不仅浪费水资源，污染环境，还会致使虾苗产生应激。循环水养殖系统模式被认为是一种环境友好的养殖模式，但其投资、运营成本及技术要求高，大面积推广难度较大。生物絮团技术通过添加有机碳源，调节C/N比，提高水中异养菌数量，利用微生物同化无机氮，絮体被对虾二次摄食，达到调控水质、促进营养物质循环、降低饲料系数的目的，该技术虽然换水量较小，但是也存在缺陷：(1)技术操作和管理过程较为复杂，在实 际生产中稳定控制难度较大；(2)采用投加有机碳源促进异养菌生长，对硝化菌群生长不利，如果调控不当，会造成氨和亚硝酸盐积累；(3)可能导致弧菌爆发和絮体积累。
生物膜法主要通过向系统中投加生物膜载体，让硝化功能微生物附着生长，不仅可以有效去除含氮化合物，尤其是高毒性的氨和亚硝酸盐，提高对虾的存活率和生长率，而且形成的生物膜可作为虾的额外食物来源，提供必要营养素，如不饱和脂肪酸、氨基酸和维生素等。
硝化功能微生物主要包括氨氧化微生物(氨氧化细菌AOB和氨氧化古菌AOA)和亚硝酸盐氧化菌(NOB)，AOB和AOA将氨转化为亚硝酸盐，NOB则进一步将亚硝酸盐转化为低毒性的硝酸盐。由于AOB、AOA和NOB属自养型微生物，代时长，繁殖速度慢、对环境因子变化敏感，使其在与异养菌竞争中处于劣势。如果将生物膜填料直接投加到养殖(标苗)池后，在其挂膜过程中水质会出现规律性变化。首先，养殖(标苗)池中存在一定数量的异养菌、氨氧化微生物和亚硝酸盐氧化菌，投加营养后，异养菌逐渐活跃起来，在吸收转化有机物同时，释放出氨，氨氧化微生物再将氨转化为亚硝酸盐。由于这两类微生物的繁殖速率相差极大，氨氧化微生物世代时间为24～36小时，而异养菌仅约20分钟，因而使其初期数量过于悬殊。随着异养菌繁殖，氨浓度也不断增加，当氨含量超出氨氧化微生物吸收消耗范围之外，氨在水体中迅速累积下来，并逐渐升高达到峰值。其后，随着氨氧化微生物不断增殖，氨氧化作用逐渐增强，氨浓度开始逐渐下降，随之亚硝酸盐浓度开始逐渐升高，亚硝酸盐氧化细菌开始发挥作用。与氨氧化细菌相比，亚硝酸盐氧化细菌代时更长，因此亚硝酸盐的转化更为缓慢，导致亚硝酸盐逐渐积累并出现峰值。由于标苗时间一般为20～30天，加上虾苗密度大，依靠直接投加填料自然挂膜过程会在标苗后期出现亚硝酸盐持续升高现象，导致虾苗生病或死亡。
发明内容
本发明的目的是为了克服现有凡纳滨对虾标粗方法中存在的水质调控难度大，虾苗成活率低、生长缓慢等问题，在充分考虑凡纳滨对虾生活习性、养殖池空间和硝化功 能微生物特性的基础上， 提供一种聚氨酯生物膜及利用其进行凡纳滨对虾高密度标苗和养殖的方法，具有无需外加有机碳源，水质稳定性好，防止致病菌侵入，减少对虾发病率，可根据污染物浓度增减填料袋数量，使用方便等优点。
为了实现上述目的，本发明通过如下技术方案实现：
一种聚氨酯生物膜，其可用于凡纳滨对虾高密度标苗和养殖，聚氨酯生物膜以聚氨酯泡沫作为生物膜载体，其上附着有氨氧化功能微生物和亚硝酸盐氧化细菌形成生物膜，其亚硝酸盐氧化速率>1.0mg/g·h，并且亚硝酸盐氧化速率与氨氧化速率比值为4～8。
本发明的一个或多个实施例中，聚氨酯生物膜以聚氨酯泡沫作为生物膜载体，通过间歇曝气、能源物质供给调控氨氧化功能微生物和亚硝酸盐氧化细菌的增殖过程，预先形成具有较高硝化活性的生物膜，其亚硝酸盐氧化速率>1.0mg/g·h，并且亚硝酸盐氧化速率与氨氧化速率比值为4～8。
本发明具体的实施例中，氨氧化微生物包括氨氧化细菌AOB和氨氧化古菌AOA，AOB和AOA可以将氨转化为亚硝酸盐，而亚硝酸盐氧化菌(NOB)则进一步将亚硝酸盐转化为低毒性的硝酸盐。氨氧化功能微生物和亚硝酸盐氧化细菌均可以选择现有报道过的菌种，只要能够实现其功能即可。本发明具体的实施例中，选用青岛海益生环境科技有限公司制备的硝化细菌制剂，当然在本发明的内容下，也可以选择类似的菌株或菌剂。
本发明实施方式中，聚氨酯泡沫，是异氰酸酯和羟基化合物经聚合发泡形成的一类高分子聚合物。
本发明的一个或多个实施例中，聚氨酯生物膜的制备方法为将聚氨酯泡沫置于人工海水中，加入硝化细菌制剂(含氨氧化功能微生物和亚硝酸盐氧化细菌)，调控其增殖过程，形成生物膜，达到亚硝酸盐氧化速率>1.0mg/g·h，并且亚硝酸盐氧化速率与氨氧化速率比值为4～8。优选的实施方案中，聚氨酯泡沫相较于人工海水按20％～40％(V/V)添加，该比例条件下，更为便利的发挥泡沫多孔材料的吸附特性，有利于提高生物膜的附着量和附着效率。
本发明的一个具体实施方式中，加入硝化细菌后调控增殖过程的方法为：按50～300毫克/升亚硝氮浓度添加亚硝酸钠，每隔12小时测定亚硝氮浓度，当亚硝氮浓度低于 0.5mg/L时，再次加入亚硝酸钠，累计添加4～6次，然后按照20～50毫克/升浓度加入氯化铵，每隔12小时测定氨氮浓度，当氨氮浓度小于0.1mg/L时，再次加入氯化铵，累计添加1～2次，即完成聚氨酯生物膜的培养过程。通过该具体操作过程，能够很好地调控聚氨酯生物膜中氨氧化微生物和亚硝酸盐氧化菌的组成，并且可以快速有效的使生物膜达到亚硝酸盐氧化速率>1.0mg/g·h，并且亚硝酸盐氧化速率与氨氧化速率比值为4～8。
本发明的一个或多个实施例中，聚氨酯生物膜的制备方法中加入硝化细菌制剂后，还包括加入微量元素液和酵母膏，以满足硝化细菌(含氨氧化功能微生物和亚硝酸盐氧化细菌)的增殖生长；优选的实施方案中，微量元素液的组成为：硫酸锌0.287mg，硫酸铜7.5mg，氯化锰14.85mg，三氯化铁5.38g，钼酸钠6.8mg，乙二胺四乙酸二钠2.4mg，六水氯化钴12mg，硫酸镍2.4mg，蒸馏水1000ml。
本发明优选的实施例中，聚氨酯生物膜的制备方法中，人工海水盐度范围为3‰～45‰，硝化细菌制剂的加入量为1％～3％(v/v)，微量元素液的加入量为0.1％(v/v)，酵母膏加入量为0.01％(w/v)。更为优选的实施例中，人工海水盐度范围为15‰～30‰。本发明实施例中，优选的采用海水素配置人工海水。
本发明的一个优选的实施例中，聚氨酯生物膜的制备方法中采用间歇曝气的方式形成生物膜。相对于溶解氧控制和自然挂膜方法，间歇曝气的方式下，聚氨酯完成生物膜挂膜的时间最快(22天)，且最终AOR和NOR值(氨氧化率(AOR)和亚硝酸盐氧化率(NOR))最高。其中一个实施例中具体的操作过程为：每隔2～4小时停止曝气15～30分钟，期间温度和溶解氧(DO)分别控制在28～30℃和4～6mg/L，时间持续20～25天。
另外一方面，本发明还公开了一种基于聚氨酯生物膜的凡纳滨对虾高密度标苗方法，该方法包括：制备如上的聚氨酯生物膜，和凡纳滨对虾标苗；通过聚氨酯生物膜对标苗过程中产生的氨和亚硝酸盐及时转化，降低氨和亚硝酸盐积累，实现凡纳滨对虾高密度标粗。
本发明的一个或多个实施例中，凡纳滨对虾标苗过程中，通过网袋将聚氨酯生物膜(即挂膜聚氨酯泡沫)悬挂在凡纳滨对虾标苗池中，并保持浸没状态。网袋的孔径以小于聚氨酯泡沫载体的粒径为准。优选的，聚氨酯生物膜投加量为标苗水体的0.5％～2.5％ (V/V)，标苗过程中，标苗池中异养菌、氨氧化微生物、亚硝酸盐氧化菌数量达到平衡，该方法无需外加有机碳源，增强水质稳定性，减少弧菌数量，促进虾苗成活率。所述网袋的材质可以选择如涤纶等耐侵蚀性高的材料。例如将预先挂膜的聚氨酯泡沫装填在涤纶材质网袋中，网袋大小为20～60升，网孔大小2～5mm，网袋带有拉链开口以方便填料的装入和取出。
本发明优选的实施例中，按照聚氨酯生物膜/人工海水为0.5～2.5％(V/V)在养殖池中放置网袋，更为优选的实施例中，在标苗池中放置网袋同时在袋子中加入1个气石补充溶解氧，来提升硝化效率。
本发明优选的实施方案中，标苗期间控制温度28～30℃，溶解氧>6.0mg/L，氨氮<0.1mg/L，亚硝氮<0.5mg/L。
本发明的一个或多个实施例中，凡纳滨对虾标苗过程中，按照密度1～3万尾/立方米水体投放凡纳滨对虾P5～P10苗，进行标苗过程管理；整个标苗过程为15～30天，可将凡纳滨对虾淡化至3‰盐度以下。出苗率可达80％以上，规格1.0～2.5厘米，所获苗种均健壮活泼、肌肉充实、肠道饱满，全身光洁无附着物，对外来刺激反应敏感，有顶水游动和沾壁行为。具体的，P5、P8、P10等为根据虾苗成色、养殖地、水源环境等进行的评级，数字越大代表质量越高。
凡纳滨对虾标苗过程中，凡纳滨对虾苗种投放应注意，按1～3万尾/立方水体投放P5～P10虾苗，如苗袋水温与标苗池差异较大，不应直接把苗倒入标苗池，一般情况下苗袋与标苗池水温每差1℃缓苗时间应增加0.5小时，如水温相差不到1℃，可直接倒入。
此外，标苗过程管理，可采用的方式为：淡化初期(1～3天),每天投喂卤虫3次,投喂时间为9:00、15:00、21:00，投喂量按照2g/1万尾(孵化率95％)，3天后除加蠕虫外，按10万尾虾苗每天投喂200-500g/天量投加虾料，5次/天投喂；再过3天后改为只投喂虾料，投料过程中应注意尽量远离填料袋，自投苗第3天开始淡化，每天盐度变化不高于1‰～3‰，每天上午添加淡水5～10厘米，3～5天后改为每天上午排水10～20厘米，接着加淡水5～10厘米，下午再加淡水5～10厘米，维持水位不变，盐度小于5‰后，每天上午排水20～30厘米接着添加淡水10～15厘米，下午添加10～15厘米，淡化至 所需盐度，总计淡化15～30天。应当明了，凡纳滨对虾标苗是对3～10日龄仔虾进行促壮淡化培育的过程，在本发明基本构思的前提下，具体的标苗过程管理也可采用些许变化方式，如投喂时间、淡化梯度等，不局限于本发明实施例方式。
标苗过程中，进行的日常检测指标包括：水温、盐度、pH、溶解氧、氨氮、亚硝氮等，以便于对标苗及时管控。
本发明的一个或多个实施方式中，标苗过程中聚氨酯生物膜可重复使用。例如，本发明的一个具体实施例中，在标苗时每隔20～30天，将聚氨酯生物膜(挂膜聚氨酯泡沫填料)从网袋取出，用同盐度的清水冲洗，去除附着的悬浮物，然后将填料填袋后重新放入标苗池使用。
本发明的一个或多个实施方式中，标苗过程在养殖池(桶)中实现，优选的，为便于控温和曝气，标苗池配备控温、曝气装置。在本发明的一个具体实施例中，标苗池由帆布围成，池内安装两排气石，池内充填人工海水；将聚氨酯生物膜置于涤纶网袋中，将网袋放置在标苗池中间，并保持浸没状态，网袋中加入气石。例如，标苗池的规格为0.2～60立方米，如2立方米、10立方米、30立方米等。优选的实施例中，标苗池的规格为10-20立方米，可以更为有效的提高虾苗成活率，降低应激性。
此外，本发明还公开了一种基于聚氨酯生物膜的凡纳滨对虾养殖方法，所述方法制备如上的聚氨酯生物膜，和在凡纳滨对虾养殖池中使用聚氨酯生物膜去除氨氮、亚硝基氮的步骤。
本发明的一个或多个实施方式中，养殖池加海水素使盐度为0.5‰-30‰，养殖池采用穿孔曝气管增氧，控制溶解氧7.0～8.5毫克/升；水温控制在28±1℃，聚氨酯生物膜(挂膜聚氨酯泡沫)装填在填料袋中浸入水中，养殖过程中每7～10天排污一次，同时补充因蒸发和排污损失的水量。定期检测养殖池中的水质变化，主要检测指标包括：温度、pH、盐度、溶解氧、氨氮、亚硝酸盐氮、硝酸盐氮等。
通过本发明的养殖方式，在养殖过程中水体氨氮、亚硝氮始终处于较低水平，氨氮小于0.1毫克/升，亚硝氮小于0.5毫克/升，经过96天的养殖，对虾成活率81.2％，饵料系数分别为1.38。
本发明针对凡纳滨对虾标苗过程的水质要求，利用氨氧化功能微生物和亚硝酸盐氧化细菌具有附着生长的特性，采用吸附的方法固定氨氧化功能微生物和亚硝酸盐氧化细菌，并通过间歇曝气、能源物质供给调控氨氧化功能微生物和亚硝酸盐氧化细菌的增殖过程，使一定数量且比例适当的氨氧化微生物和亚硝酸盐氧化菌在聚氨酯上生长形成稳定生物膜，并确定了聚氨酯生物膜氨氧化速率和亚硝酸盐氧化速率的大小和比值。然后将挂膜聚氨酯用网袋悬挂在凡纳滨对虾标苗池中，使标苗池中异养菌、氨氧化微生物、亚硝酸盐氧化菌数量达到平衡，从而在标苗过程中产生的氨和亚硝酸盐及时得到转化，不会出现氨和亚硝酸盐积累，实现凡纳滨对虾高密度标粗。该方法无需外加有机碳源，具有水质稳定性好，弧菌数量低，虾苗成活率高，可根据污染物浓度增减填料袋数量，使用方便等特点，特别适用于内陆地区高密度凡纳滨对虾标苗。
附图说明
图1聚氨酯泡沫挂膜前后及使用后表面形态变化(边长为1-2cm的立方体)；(A)挂膜前；(B)挂膜后；(C)使用10天后，(D)使用20天后。
图2聚氨酯泡沫挂膜后氨氧化率(AOR)和亚硝酸盐氧化率(NOR)随时间变化情况
图3为本发明涉及实施例2标苗池的结构图；1横梁，2水面，3填料袋，4气石，5阀门，6排水管。
图4为本发明涉及实施例2标苗过程中氨氮和亚硝氮浓度变化情况。
具体实施方式
以下结合具体实施例，对本发明进行详细说明。
下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1：
聚氨酯泡沫挂膜可以通过以下步骤实现：
将0.04m 3聚氨酯泡沫(规格：2厘米)清洗后置于含0.1m 3人工海水的塑料桶中，人工海水盐度为30‰，加入2％(v/v)硝化细菌制剂(青岛海益生环境科技有限公司)， 0.1％(v/v)微量元素液和0.01％(w/v)酵母膏。微量元素液组成见表1。
表1微量元素液配方
Figure PCTCN2020079551-appb-000001
在生物膜培养过程中，共分5次添加亚硝酸钠400g，每次添加80g。当亚硝氮浓度降至0.5mg/L以下时，再次加入亚硝酸钠；加完5次亚硝酸钠之后，分2次添加60g氯化铵，每次添加30克，当氨氮浓度小于0.5mg/L时，即完成聚氨酯生物膜的培养过程，在整个培养过程中，每隔2小时停止曝气15分钟，期间温度和溶解氧(DO)分别保持在28±1.0℃和4.5～6.0mg/L。
挂膜结束后，随机取8个聚氨酯泡沫单体，分成两组，加入到2个含有300毫升人工海水、0.1％(v/v)微量元素溶和0.01％(w/v)酵母膏的500毫升锥形瓶中，分别加入亚硝酸钠和氯化铵使氨氮和亚硝氮初始浓度为50mg/L和200mg/L，然后在150转/分，28℃条件下摇瓶培养，每12小时测量一次氨氮和亚硝酸盐氮浓度，按以下公式计算氨氧化率(AOR)和亚硝酸盐氧化率(NOR)。AOR或NOR＝K·V/W，其中K是氨氮或亚硝氮浓度(mg/L)散点图的斜率，V为溶液体积，W为4个氨酯泡沫单体的干重(g)，最终测定AOR为0.250mg/g·h，NOR为1.475mg/g·h，NOR与AOR比值为5.9。
采用荧光原位杂交方法(FISH)分析聚氨酯生物膜中氨氧化细菌(AOB)和亚硝酸 盐氧化细菌(NOB)分布情况，其中AOB采用NS0190探针，序列为5’-CGATCCCTGCTTTTCTCC-3’，探针5’-端用FAM标记；NOB采用NIT3探针，序列为5’-5CCTGTGCTCCATGCTCCG-3’，探针5’-端用HEX标记，竞争性探针CNIT3序列为5’-CCTGTGCTCCAGGCTCCG-3’。检测结果见表2，聚氨酯生物膜中AOB丰度为24.8％，NOB丰度为72.7％(其他比例由于氨氧化细菌的增殖速率快，会在标苗过程中出现亚硝酸盐峰值，从而对虾苗产生毒害)。表明采用本发明方法能够很好地调控聚氨酯生物膜中氨氧化微生物和亚硝酸盐氧化菌的组成。
表2聚氨酯生物膜FISH检测
Figure PCTCN2020079551-appb-000002
为了验证聚氨酯填料生物膜培养过程中亚硝酸钠和氯化铵投加比例的有效性，分别采用不同亚硝酸钠和氯化铵投加量培养聚氨酯填料生物膜，采用FISH方法分析其氨氧化菌和亚硝酸盐氧化菌丰度，挂膜结束后，每组随机取4个聚氨酯泡沫单体，加入到含有300毫升人工海水、0.1％(v/v)微量元素溶和0.01％(w/v)酵母膏的500毫升锥形瓶中，加入氯化铵使氨氮初始浓度为5mg/L，然后在150转/分，28℃条件下摇瓶培养，每4小时测量一次氨氮和亚硝酸盐氮浓度，确定实验过程中亚硝氮峰值浓度。实验结果见表3。从表中可以看出，采用亚硝酸钠/氯化铵为400g/60g投加比例，其亚硝酸盐氮峰值浓度最低，仅为0.06mg/L，而其他实验组亚硝酸盐氮峰值浓度都比较高，且随着氯化铵投加量的增加，AOB的丰度也越高，相应亚硝酸氮峰值浓度也越高。
表3不同亚硝酸钠和氯化铵投加量制备聚氨酯生物膜对氨氮的转化性能
Figure PCTCN2020079551-appb-000003
Figure PCTCN2020079551-appb-000004
为了验证采用上述聚氨酯泡沫挂膜方法的有效性，比较分析了不同挂膜方法的差异，分别采取溶解氧控制、间歇曝气、自然挂膜方法，实验在有效体积为20升的玻璃缸进行，玻璃缸内装有用海水素配制盐度30‰人工海水20升，实验组1～6号加入2％(v/v)硝化细菌制剂(青岛海益生环境科技有限公司)，0.1％(v/v)微量元素液和0.01％(w/v)酵母膏。用实验组7号采用自然挂膜方式，在玻璃缸中加入1.5克对虾饵料。采用加热棒控制水温28℃。将30％(V/V)聚氨酯清洗后浸没在水面之下。然后实验过程参考上述方法，共分5次添加亚硝酸钠80g，每次添加16g。当亚硝氮浓度降至0.5mg/L以下时，再次加入亚硝酸钠；加完5次亚硝酸钠之后，分2次添加12g氯化铵，每次添加6克，当氨氮浓度小于0.5mg/L时，即完成聚氨酯生物膜的培养过程，具体控制指标和实验结果见表4。在每隔2小时停止曝气15分钟的间歇曝气条件下，聚氨酯完成挂膜的时间最快(22天)，且最终AOR和BOR值最高。
表4 7种聚氨酯挂膜方法的控制指标和实验结果
Figure PCTCN2020079551-appb-000005
Figure PCTCN2020079551-appb-000006
将制成聚氨酯生物膜活性填料用塑料密封袋密封保存，每隔1个月测定氨氧化率(AOR)和亚硝酸盐氧化率(NOR)变化情况，结果见图2，从图2中可以看出经过1年时间，其氨氧化率(AOR)和亚硝酸盐氧化率(NOR)仍能维持较高活性，分别相当于初始值的48％和55％。
实施例2：
一种基于聚氨酯生物膜的凡纳滨对虾高密度标苗方法
标苗池由帆布材料制成，有效容积2.0m 3(长：2.0m，宽：1.0m，高：1.2m)。池内安装两排共16个气石。池内开始时充填0.6m高人工海水，含盐量18‰。将0.04m 3预先挂膜的聚氨酯泡沫装在两个网孔大小为2mm的涤纶网袋中，将网袋放置在标苗池中间，并保持浸没状态，每个网袋中加入一个气石。
将3万尾P8凡纳滨对虾虾苗投放到标苗池，初期(1～3天),每天投喂卤虫3次,投喂时间为9:00、15:00、21:00，投喂量按照2g/1万尾(孵化率95％)；3天后除加蠕虫外，按10万尾虾苗每天投喂200-500g/天量投加虾料，5次/天投喂；再过3天后改为只投喂虾料。投料过程中应注意尽量远离填料袋。标苗过程中，每天对虾苗生长和剩余饵料进行评估，并用于指导饲料配给。
自投苗第3天开始淡化，每天上午添加淡水10厘米，到第6天，水位升至100厘米，然后每天上午排水10厘米，接着加淡水5厘米，下午再加淡水5厘米，维持水位不变。盐度小于5‰后，每天上午排水20厘米接着添加淡水10厘米，下午添加10厘米。到第18天时，盐度降至2.9，继续养殖到第25天开始分苗。虾苗平均体长2.1cm，平均体重0.18克，成活率为85.2％。
在标苗过程中养殖水体氨氮浓度始终低于0.1毫克/升，亚硝氮浓度在前12天低于0.2毫克/升，之后有所上升，但始终低于0.5毫克/升；硝氮呈逐渐升高趋势，从2.1毫克/升增至8.3毫克/升，pH则由7.83降至7.35。
实施例3：
一种基于聚氨酯生物膜的凡纳滨对虾高密度标苗方法
为验证一种基于聚氨酯生物膜的凡纳滨对虾高密度标苗方法的有效性，选取2个水泥池进行验证，养殖池大小为4.0米×3.0米×1.2米，水深1.0米，水体体积为12.0立方米，用海水晶调节盐度为18‰，实验期间控制温度28℃，溶解氧7.0-8.5毫克/升；养殖池I加入3个挂膜聚氨酯填料网袋，网袋孔径2毫米，总体积为0.12立方米，占养殖水体总体积的1％。养殖池II不添加聚氨酯填料，只依靠换水净化水质。
分别将15万尾P5虾苗投放到两个标苗池，饵料投喂方法同实施例2。自投苗第3天开始淡化，标苗池I淡化方法与实施例2方法相同，其在整个标苗过程中其水质变化情况与实施例2也基本相同，氨氮始终在0.1毫克/升以下，亚硝氮在0.4毫克/升以下。标苗池II在前5天水质较为稳定，之后亚硝氮开始升高并在第8天的时候升至0.5毫克/升，之后不得不每天换水以保证亚硝氮在0.5毫克/升以下，到第15天每天换水量逐渐提高到60％～80％，对虾应激现象比严重。
标苗池I养殖到第25天开始分苗。虾苗平均体长2.1cm，平均体重0.18克，成活率为86.4％。标苗池II由于后期换水量大，对虾应激严重，只养殖到16天即结束标苗过程，虾苗平均体长1.1cm，平均体重0.12克，成活率为55.7％。
实施例4：
本实施例进一步验证聚氨酯生物膜在凡纳滨对虾养殖中的使用效果。选用1个水体体积为20立方米的水泥池进行凡纳滨对虾养殖试验，养殖池大小为5.0米×4.0米×1.2米，水深1.0米。将地下水经过沉淀、过滤处理后注入养殖池，加海水素使盐度为3‰，养殖池采用穿孔曝气管增氧，溶解氧7.0～8.5毫克/升。水温控制在28±1℃，养殖前期(1～2月)将按照实施例1方法制备挂膜聚氨酯泡沫0.1立方米装填在两个填料袋中，填料袋网孔为2毫米，养殖后期(3～4月)将挂膜聚氨酯泡沫体积增至0.2立方米。养殖过程中每7～10天排污一次，同时补充因蒸发和排污损失的水量。
投放按照实施例2方法标粗淡化的凡纳滨对虾2万尾(密度1000尾/立方米)，养殖过程中按常规方法投饵和水质管理，养殖期间每隔20天左右可以将填料包取出，在同盐度清水中清洗去除吸附的颗粒物，实现聚氨酯填料的吸附能力再生。
定期检测养殖池中的水质变化，主要检测指标包括：温度、pH、盐度、溶解氧、氨氮、亚硝酸盐氮、硝酸盐氮等。在养殖过程中水体氨氮、亚硝氮始终处于较低水平，氨氮小于0.1毫克/升，亚硝氮小于0.5毫克/升，经过96天的养殖，对虾成活率81.2％，饵料系数分别为1.38。
一个养殖周期结束后，采用清水将填料包冲洗干净，集中放置在1个养殖池中保存，保存条件：水体溶解氧4.0mg/L以上，到下一个养殖周期将填料袋重新悬挂在养殖池内使用。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Figure PCTCN2020079551-appb-000007
Figure PCTCN2020079551-appb-000008

Claims (10)

  1. 一种聚氨酯生物膜，其可用于凡纳滨对虾高密度标苗和养殖，其特征在于，聚氨酯生物膜以聚氨酯泡沫作为生物膜载体，其上附着有氨氧化功能微生物和亚硝酸盐氧化细菌形成生物膜，其亚硝酸盐氧化速率>1.0mg/g·h，并且亚硝酸盐氧化速率与氨氧化速率比值为4～8。
  2. 根据权利要求1所述的聚氨酯生物膜，其特征在于，聚氨酯生物膜的制备方法为将聚氨酯泡沫置于人工海水中，加入含氨氧化功能微生物和亚硝酸盐氧化细菌的微生物制剂，调控其增殖过程，形成生物膜；聚氨酯泡沫相较于人工海水按体积比20％～40％添加。
  3. 根据权利要求2所述的聚氨酯生物膜，其特征在于，加入微生物制剂后调控菌群增殖过程的方法为：按50～300毫克/升亚硝氮浓度添加亚硝酸钠，每隔12小时测定亚硝氮浓度，当亚硝氮浓度低于0.5mg/L时，再次加入亚硝酸钠，累计添加4～6次，然后按照20～50毫克/升浓度加入氯化铵，每隔12小时测定氨氮浓度，当氨氮浓度小于0.1mg/L时，再次加入氯化铵，累计添加1～2次，即完成聚氨酯生物膜的培养过程。
  4. 根据权利要求2或3任一项所述的聚氨酯生物膜，其特征在于，聚氨酯生物膜的制备方法中，人工海水盐度范围为3‰～45‰，硝化细菌制剂的加入量为1％～3％，还加入微量元素液和酵母膏，微量元素液的加入量为0.1％，酵母膏加入量为0.01％。
  5. 根据权利要求1-3任一项所述的聚氨酯生物膜，其特征在于，聚氨酯生物膜的制备方法中采用间歇曝气的方式形成生物膜。
  6. 根据权利要求5所述的聚氨酯生物膜，其特征在于，间歇曝气，每隔2～4小时停止曝气15～30分钟，期间温度和溶解氧分别控制在28～30℃和4～6mg/L，时间持续20～25天。
  7. 一种基于聚氨酯生物膜的凡纳滨对虾高密度标苗方法，其特征在于，包括：制备如权利要求1-6任一项所述的聚氨酯生物膜，和进行凡纳滨对虾标苗过程。
  8. 根据权利要求7所述的凡纳滨对虾高密度标苗方法，其特征在于，凡纳滨对虾标苗过程中，通过网袋将聚氨酯生物膜悬挂在凡纳滨对虾标苗池中，并保持浸没状态； 聚氨酯生物膜投加量为标苗水体的0.5％～2.5％；标苗过程在配备控温、曝气装置以及悬挂填料袋横梁的养殖池(桶)实现。
  9. 根据权利要求8所述的凡纳滨对虾高密度标苗方法，其特征在于，标苗期间控制温度28～30℃，溶解氧>6.0mg/L，氨氮<0.1mg/L，亚硝氮<0.5mg/L；
    按照密度1～3万尾/立方米水体投放凡纳滨对虾P5～P10苗，进行标苗过程管理；
    标苗过程管理：淡化初期,每天投喂卤虫3次,投喂时间为9:00、15:00、21:00，投喂量按照2g/1万尾，3天后除加蠕虫外，按10万尾虾苗每天投喂200-500g/天量投加虾料，5次/天投喂；再过3天后改为只投喂虾料，自投苗第3天开始淡化，每天盐度变化不高于1‰～3‰，每天上午添加淡水5～10厘米，3～5天后改为每天上午排水10～20厘米，接着加淡水5～10厘米，下午再加淡水5～10厘米，维持水位不变，盐度小于5‰后，每天上午排水20～30厘米接着添加淡水10～15厘米，下午添加10～15厘米，淡化至所需盐度，总计淡化15～30天。
  10. 一种基于聚氨酯生物膜的凡纳滨对虾养殖方法，其特征在于，所述方法包括制备如权利要求1-6任一项所述的聚氨酯生物膜，和和在凡纳滨对虾养殖池中使用聚氨酯生物膜去除氨氮、亚硝基氮的步骤；
    包括养殖池加海水素使盐度为0.5‰-30‰，养殖池采用穿孔曝气管增氧，控制溶解氧7.0～8.5毫克/升；水温控制在28±1℃，聚氨酯生物膜装填在填料袋中浸入水中，养殖过程中每7～10天排污一次，同时补充因蒸发和排污损失的水量。
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