CN110468066B

CN110468066B - Aerobic denitrifying strain and application thereof

Info

Publication number: CN110468066B
Application number: CN201910689536.1A
Authority: CN
Inventors: 张宏耀; 杨青
Original assignee: China Complete Equipment Import And Export Group Co ltd; China National Complete Plant Import & Export Corp ltd
Current assignee: China complete equipment import and Export Group Co.,Ltd.
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2021-09-17
Anticipated expiration: 2039-07-29
Also published as: CN110468066A

Abstract

The invention relates to Pseudomonas (Pseudomonas sp.N200), which has a deposit number of: CGMCC No. 18072. The pseudomonas can be subjected to efficient denitrification under the condition of high dissolved oxygen.

Description

Aerobic denitrifying strain and application thereof

Technical Field

The invention belongs to the technical field of microbial environment protection, and particularly relates to an aerobic denitrifying strain pseudomonas N200 and application thereof in denitrification.

Background

Nitrate Nitrogen (NO)₃ ^-N) is a very extensive nitrogen form occurring in nature, and is one of the important forms of nitrogen element existing in the environment. NO in aqueous environment₃ ^-N pollution, mainly resulting from agricultural irrigation processes and from urban runoff, hygiene and irregularities in the disposal of industrial waste, leaks of infected systems, etc.

At present, NO of the pollution level of mu M to mM can be detected in rivers, lakes and the like as natural water bodies₃ ^--N residues. For NO₃ ^-N, wastewater treatment plants (plants) are removing ammonia Nitrogen (NH)₄ ⁺N) while easily causing NO₃ ^--N and NO₂ ^-Accumulation of N, but usually NO₃ ^-N is not used as a standard for wastewater discharge, so this fraction of NO₃ ^-N will be discharged into groundwater, rivers, lakes, etc.

NO₃ ^-N causes severe eutrophication of water in rivers, lakes, etc. If nitrate nitrogen, i.e. nitrate, is present in high concentrations in drinking water, it can have an effect on human health, for example, causing methemoglobinemia in infants, andand bladder cancer, non-Hodgkin's lymphoma, ovarian cancer, digestive tract cancer in adults. Thus, water NO₃ ^-N seriously affects human health, ecology and aquatic ecosystem, and removes NO₃ ^-N is essential to control these problems.

Currently, NO is removed from water₃ ^-Common techniques for-N include: adsorption, ion exchange, electrochemistry, reverse osmosis, membrane filtration, biological denitrification and the like. Among them, the biological denitrification technology, especially the addition of microbial agents, has become one of the important means for removing water body nitrates due to the advantages of small investment, good effect, no secondary pollution and the like.

The denitrification is an important link of the biogeochemical cycle of nitrogen, and can directly remove NO₃ ^--N and N₂Is removed. The traditional denitrification is anaerobic denitrification, and the nitrification process is an aerobic reaction, so that the denitrification can not be synchronously carried out. Generally, an aerobic-anaerobic alternative process is adopted for nitrification-denitrification, but the problems of complex process, high facility construction cost, large occupied area of reaction equipment, high requirement on later-stage operation and maintenance and difficult application feasibility of a watershed environment exist. However, with the first isolation of the first aerobic denitrifying bacteria in 1980, there is increasing evidence that aerobic denitrifying bacteria play a non-negligible role in the nitrogen cycle of the ecosystem. The aerobic denitrification process has low requirements on process equipment, saves cost, can greatly improve denitrification efficiency, has high application value and prospect, and has wide application space. In addition, the aerobic denitrification can be applied to biological sewage treatment and can also be applied to in-situ remediation of rivers and lakes.

The aerobic denitrifying bacteria are obligate or facultative aerobic bacteria, and have low content in nature, so the aerobic denitrifying bacteria are difficult to become dominant strains in natural environment, and the separation of the aerobic denitrifying bacteria has great difficulty. Therefore, the separation and culture of the aerobic denitrification strains with strong living capacity and high denitrification function and the expansion of the existing aerobic denitrification strain bank provide important material basis for the future application of the aerobic denitrification strains.

The high-efficiency aerobic denitrifying bacteria pseudomonas N200 can be directly applied to treatment of domestic sewage in cities and rural areas and in-situ ecological restoration of rivers and lakes, and has a good denitrification effect. By adding the bacterium, the removal efficiency of nitrogen in the water body can be effectively improved.

Disclosure of Invention

The invention aims to provide an aerobic denitrifying strain pseudomonas N200 and application thereof in sewage denitrification and river and lake remediation. The strain has good denitrification effect, and can be applied to sewage treatment, in particular to sewage treatment with high nitrate nitrogen content.

The aerobic denitrifying strain pseudomonas N200 is obtained by separating and screening from a white lake culture cofferdam, and is classified and named as: pseudomonas sp.N200, a new species, which is preserved in China general microbiological culture Collection center (CGMCC; address No. 3 Xilu-1. Beijing, Chaoyang district, Beijing) in 7.4.7.2019, with the preservation number: CGMCC No. 18072. The 16S rDNA sequence of the aerobic denitrifying strain is shown as Seq ID: 1, the length is 1459 bp.

As shown in fig. 1, pseudomonas N200 has the following phenotypic characteristics: after the bacterial colony grows for 2 days at 28 ℃ on 1/10LB culture medium and denitrifying agar culture medium, the bacterial colony is a light yellow and round bacterial colony with the diameter of 1-2mm, the surface of the bacterial colony is smooth and moist, and the edge of the bacterial colony is in a mist shape, has no halo and does not bulge.

As shown in FIG. 2, the individual cells are short rods with a size of 1.0-1.3 μm × 0.40-0.45 μm and are arranged singly or in pairs. The physiological and biochemical characteristics are as follows: is gram negative. The growth conditions of the aerobic denitrification strains are as follows: the temperature is 15-42 ℃, and the pH is 6-10.

Determination of species

The model strains most similar to the aerobic denitrification strains in sequence are as follows: the Pseudomonas composi CCUG 59231(T) (NCBI accession No.: NR _116992) had a 16S full-length similarity of 97.94%. A variety of Pseudomonas model strain 16S sequences similar to the above were selected for tree construction, and a Pseudomonas denitificates strain X2(T) belonging to the genus Pseudomonas was selected as an outer root, and a tree was constructed by the NJ method using the software MEGA6.0, in which Bootstrap selection was 1000. As shown in FIG. 3, it was confirmed from the sequence similarity and the evolutionary relationship that the aerobic denitrifying strain of the present invention is a member of the genus Pseudomonas and is a new strain.

Performing whole genome sequencing on the aerobic denitrifying strain, and selecting a whole genome (NCBI accession number: NZ _ FOWP00000000) of a model strain Pseudomonas composition CCUG 59231(T) with the most similar sequence for DAN-DNA hybridization, wherein the estimated value is 23.30 percent and is far less than a threshold value (70 percent); the difference in the G + C content was 3.40.

The pseudomonas N200 has a high-efficiency denitrification function. By culturing and activating under the proper condition that the temperature is 28 ℃ and the dissolved oxygen is more than 3mg/L, preferably 3-7mg/L, more preferably 5-7mg/L, and coexisting with the water body for a period of time, the removal rate of the nitrate nitrogen in the water body can reach 100 percent, and the total nitrogen removal rate can reach more than 50 percent.

In one embodiment, the test sample of sewage is selected from rural domestic sewage discharged into the white ocean lake. The aerobic denitrifying strain pseudomonas N200 is indigenous microorganisms screened from the lake area, so that the aerobic denitrifying strain pseudomonas N200 has the advantages of considerable economic benefit, great saving of sewage treatment cost and high strain safety when being used for purifying rural domestic sewage discharged into the lake area.

The aerobic denitrifying strain pseudomonas N200 (hereinafter, referred to as the strain of the invention or pseudomonas N200) can be denitrified by denitrification, so that the aerobic denitrifying strain pseudomonas N200 is further suitable for treating various nitrate nitrogen polluted water bodies in other regions of China.

Drawings

FIG. 1 colony morphology of the aerobic denitrifying strain Pseudomonas N200 on a plate.

FIG. 2 shows the cell morphology of the aerobic denitrifying bacterium Pseudomonas N200 under an electron microscope.

FIG. 3 phylogenetic analysis of the aerobic denitrifying bacteria Pseudomonas N200 and similar model strains

FIG. 4 Nitrogen elimination Effect of Pseudomonas N200, A is nitrogen elimination in denitrifying medium, B is inOD in the denitrifying medium₆₀₀Value-reflected proliferation curve, wherein NO₃ ^--N represents nitrate nitrogen; NO₂ ^--N represents nitrous nitrogen; TN means total nitrogen.

Detailed Description

In one embodiment of the present invention, a single colony of pseudomonas N200 is picked up, cultured in a medium (containing 5% o tryptone, 2.5% o yeast extract powder, 1% o glucose, 1% o soluble starch, 0.5% o monopotassium phosphate, 0.5% o disodium hydrogen phosphate, 0.2% o magnesium sulfate, pH 7.3) at a dissolved oxygen of 3mg/L, preferably 3 to 7mg/L, more preferably 5 to 7mg/L, and at a temperature of 28 ℃ for 24 hours, and then inoculated into nitrogen-containing sewage at a ratio of 1/100 (mass/volume ratio) based on the volume of the nitrogen-containing sewage for 48 hours.

The composition of the aerobic denitrification culture medium is as follows: 1g/L potassium nitrate, 4.68g/L sodium succinate, 0.2g/L magnesium sulfate, 0.01g/L calcium chloride, 0.07g/L EDTA0, 0.5g/L potassium dihydrogen phosphate, 0.5g/L disodium hydrogen phosphate, 0.01g/L ferrous sulfate, 5g/L sodium chloride, a trace element solution (EDTA 57.1g/L, 3.9g/L zinc sulfate, 7g/L calcium chloride, 1g/L manganese chloride, 5g/L ferrous sulfate, 1.1g/L ammonium molybdate, 1.6g/L copper sulfate, 1.6g/L cobalt chloride, pH 6.0)2ml/L, and pH 7.0-7.5.

According to the HJ 636 + 2012 standard: the determination of Total nitrogen in water (national environmental protection standard of the people's republic of China) is carried out, wherein, under the specified conditions, Total Nitrogen (TN) refers to the sum of dissolved nitrogen in a sample and nitrogen in suspended matters which can be determined, and comprises the following steps: nitrate nitrogen, nitrite nitrogen, inorganic ammonium nitrogen, dissolved ammonia and most of the nitrogen in organic nitrogen compounds.

The content of various aqueous components in the present invention can be determined using measurement methods known to those skilled in the art, for example: the ammonia nitrogen, nitrate nitrogen and nitrite nitrogen can be measured by a Thermo Fisher Scientific water quality analyzer; total nitrogen was determined as above using HJ 636-; COD was determined using a Hash COD Rapid reaction kit (Hach, USA).

Although the bacteria preparation containing pseudomonas N200 (for example, as a liquid culture) is directly introduced into a natural river section as a water purification agent in the embodiment of the present invention, the bacteria preparation may be in the form of a frozen stock, a dry powder, a concentrated bacteria preparation diluted only at the time of use, or the like, and freeze-drying of the bacteria preparation is a conventional technique in the art, and various additives such as a stabilizer, a bacteria conserving agent, a nutrient, a taste masking agent, an osmotic pressure regulator, a colorant (for example, for visualizing the concentration of the bacteria preparation), a pH indicator, and the like may be contained in the water purification agent without affecting the effect of the present invention.

When the strain of the present invention is used for denitrification treatment in a sewage purification apparatus, the sewage purification apparatus may employ a domestic type sewage purification tank, an industrial type water purification tank for a water purification plant or a fermentation apparatus, or the like, but is not limited thereto.

Examples

Example 1: isolation and characterization of Pseudomonas N200

(1) Enrichment and isolation of aerobic denitrifying strains

Collecting bottom mud and sewage from cofferdam of lake culture, placing in sterile collection bag, culturing in microorganism-enriched culture medium containing nitrate as unique nitrogen source at 28 deg.C, and controlling dissolved oxygen content at 3-7mg/L during culture period.

Transferring every 5 days, enriching for 30 days, performing gradient dilution with sterile double distilled water, and selecting appropriate dilution concentration (10)^-5-10^-7Dilution factor) the diluted solution was applied to a solid medium such as LB, 1/10LB, and R2A (2015 "chinese pharmacopoeia"), and colonies with a single form were selected from among colonies grown on the solid medium, and further purified and cultured.

The components of the microorganism enrichment culture medium are as follows: potassium nitrate 0.7g/L, sodium succinate 5g/L, sodium acetate 2g/L, trace element liquid A (dipotassium hydrogen phosphate 5g/L, magnesium sulfate 2.5g/L, sodium chloride 2.5g/L, ferrous sulfate 0.05g/L, manganese sulfate 0.05g/L)50 ml/L.

(2) Screening of aerobic denitrifying strains

Culturing the strain obtained by separation and purification in the step (1) in an R2A liquid culture medium to the end of logarithm, then centrifugally collecting the strain, suspending the strain in an aerobic denitrification culture medium, and adjusting to enable the strain to be in suspensionOD₆₀₀The value is 1, inoculating into aerobic denitrification culture medium at a volume ratio of 1/100, culturing at 30 deg.C and 200rpm under the condition of maintaining dissolved oxygen amount at 5mg/L or more for 48 hr, and measuring NO₃ ^--N removal rate. Selecting NO therein₃ ^-Strains with N removal rate higher than 90% and total nitrogen removal rate higher than 50% are taken as candidate aerobic denitrification strains.

The aerobic denitrification culture medium comprises the following components: 1g/L potassium nitrate, 4.68g/L sodium succinate, 0.2g/L magnesium sulfate, 0.01g/L, EDTA 0.07.07 g/L calcium chloride, 0.5g/L potassium dihydrogen phosphate, 0.5g/L disodium hydrogen phosphate, 0.01g/L ferrous sulfate, 5g/L sodium chloride, and 2ml trace element solution B (the trace element solution B comprises 57.1g/L EDTA, 3.9g/L zinc sulfate, 7g/L calcium chloride, 1g/L manganese chloride, 5g/L ferrous sulfate, 1.1g/L ammonium molybdate, 1.6g/L copper sulfate, and 1.6g/L, pH 6.0.0 cobalt chloride), and the pH value is 7.0-7.5.

(3) Identification of aerobic denitrifying strains

The 16SrDNA of the candidate aerobic denitrifying bacteria strain obtained in (2) was amplified by PCR and inserted into a commercially available TaKaRa brand

In the 19-T Vector, the 16S rDNA full-length sequence of the aerobic denitrifying strain is obtained by using Vector primers (M13F: TGTAAAACGACGGCCAGT and M13R: CAGGAAACAGCTATGACC) in an Ezbiochoud databasehttp://www.ezbiocloud.net/) Is determined to be Pseudomonas sp. The total length of 16S rDNA of the aerobic denitrifying strain is 1459bp, the searched most similar model strain is Pseudomonas composti CCUG 59231(T), and the similarity of the two is 97.94%.

Downloading a 16S rDNA sequence similar to the 16SrDNA sequence of the aerobic denitrifying strain, selecting a maximum likelihood method by using MEGA6.0 software, and performing similarity repeated calculation for 1000 times to obtain the 16S rDNA phylogenetic tree of the aerobic denitrifying strain (figure 4).

The aerobic denitrifying strain is subjected to whole genome sequencing in China science and technology, and DNA-DNA hybridization analysis is carried out on the aerobic denitrifying strain and a genome of a model strain Pseudomonas composti CCUG 59231(T), so that the homology of the aerobic denitrifying strain and the genome is only 23.30 percent and is far less than the threshold value of the same species, namely 70 percent; the difference in G + C content between the two was 3.40. Therefore, the identification result shows that the aerobic denitrification strain is a new strain with an aerobic denitrification function.

Example 2: denitrification effect of pseudomonas N200 on denitrification culture medium

The aerobic denitrifying strain pseudomonas N200 is inoculated into an R2A liquid culture medium test tube as a single colony and is subjected to activation culture at 30 ℃ and shaking in a shaking table at 160 rpm.

After 24h of activation culture, according to the volume of the culture medium, transferring to NO according to the mass/volume ratio of 1/100₃ ^-And (3) culturing in a denitrification culture medium with the N content of 140mg/L for 48 hours in a culture bottle at 28 ℃ under shaking conditions of a shaking table at 180rpm, wherein the dissolved oxygen content ranges from 3mg/L to 7mg/L during the culture, and most of the time is more than 5 mg/L. Wherein samples are taken every 6h and the OD of the samples is determined at each time point₆₀₀。

After 48h incubation, all samples at each time point were centrifuged at 8000rpm for 10 minutes, and the supernatants were removed and the nitrate, nitrite and total nitrogen contents were determined separately in each supernatant. The time is plotted on the x-axis against the content of each nitrogen species on the y-axis.

In FIG. 4, NO₃ ^--N represents nitrate nitrogen; NO₂ ^--N represents nitrous nitrogen; TN means total nitrogen.

As can be seen from FIG. 4, after 48 hours, the nitrate nitrogen removal rate of the Pseudomonas sp N200 reaches 99%, the total nitrogen is reduced by more than 80%, and the accumulation of nitrite nitrogen is less than 15%. The pseudomonas N200 has strong aerobic denitrification capability, the total nitrogen removal rate reaches 80%, and the water body with the final nitrate nitrogen level as low as 0mg/L and the total nitrogen level as low as below 30mg/L can be obtained by using the pseudomonas N200 to remove nitrogen.

Example 3 short-term denitrification Effect of Pseudomonas bacteria N200 on domestic Sewage

The aerobic denitrifying strain pseudomonas N200 of the invention is treated according to the following stepsActivation and scale-up culture were carried out in the same manner as in example 2 to obtain a microbial inoculum containing Pseudomonas sp N200. OD is measured by the volume of the medium water with the mass/volume ratio of one thousandth₆₀₀Adding a pseudomonas N200-containing microbial inoculum of not less than 1m at room temperature³1m of membrane module-containing reclaimed water³In the membrane module chamber of domestic small-sized sewage purification tank. Wherein, the reclaimed water is aerated, and the dissolved oxygen is kept at 3-7mg/L in the whole denitrification process.

The resulting mixture was allowed to stand at room temperature for 5 days, during which time Pseudomonas sp.N200 was allowed to proliferate naturally and adsorbed on a carrier membrane (i.e., natural biofilm formation) provided in a purification tank. After 5 days, the reclaimed water is fed into the purification tank from the water inlet, and the speed of the reclaimed water fed into and discharged from the purification tank is 0.6m at most³The day is. The concentration of nitrate nitrogen in the reclaimed water of the inlet water is 20mg/L, the total nitrogen is 29mg/L, and the temperature of the inlet water is 20 ℃.

After 3 days of continuous water inflow, the quality of the effluent collected from the water outlet tends to be stable, and an effluent sample is collected for nitrate Nitrogen (NO)₃ ^--N, mg/L), Ammonia Nitrogen (NH)₄ ⁺-N, mg/L), nitrous Nitrogen (NO)₂ ^-N, mg/L), total nitrogen (TN, mg/L), chemical oxygen demand (COD, mg/L) were determined: ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were measured by Thermo Fisher Scientific water quality analyzer, total nitrogen was measured by HJ 636-.

The result shows that after the strain pseudomonas N200 is added, the removal rate of nitrate nitrogen in the reclaimed water reaches over 90 percent, the removal rate of total nitrogen reaches 55 percent, and the Chemical Oxygen Demand (COD) is obviously reduced.

TABLE 1 short-term denitrification Effect of Pseudomonas sp N200 on domestic Sewage

	Nitrate nitrogen (mg/L)	Total nitrogen (mg/L)	COD(mg/L)
				Before reclaimed water treatment	20.1±0.05	29±0.07	68±0.50
After the reclaimed water is treated	1.7±0.01	13±0.07	35±0.50

Example 4 short term remediation of urban rivers by Pseudomonas N200

A section of northern urban river is selected, the water replenishing source of the urban river is reclaimed water, and the average river width is 20 meters. The water body dissolves oxygen, has high transparency, serious eutrophication and overgrowth of aquatic weeds. The main reason for the excessive growth of aquatic weeds is the eutrophication of nitrogen elements caused by the high nitrate content.

Selecting a water area with slow flow rate at the upstream of the river channel, wherein the test time is early autumn, and the highest and lowest water temperature difference of the river water in the section is not more than 6-8 ℃ within 24 hours. The length of the water area is about 20m, the average depth is 1m through 5-point average depth measurement and calculation, and the total water volume (V) of the river at the section is estimated according to the average river width.

A liquid microbial inoculum (OD) containing the aerobic denitrifying bacterial strain of the present invention₆₀₀About 2, the number of viable bacteria is not less than 1 × 10¹⁰CFU/mL) is added according to the mass/volume ratio of one thousandth, wherein the total water amount (V) is measured, and when the CFU/mL) is added, the water temperature is 28 ℃ and the pH value is 8.1.

And (3) sampling 5 points uniformly distributed from the beginning to the end of the water area before the bacteria are thrown to obtain a water body sample without the microbial inoculum.

After 3 days (72 hours) of the microbial inoculum is added, 5 point samples at the same position are similarly taken to obtain a restored (denitrified) water body sample of the section of water area. Nitrate Nitrogen (NO) in the water area of the samples before the addition of the microbial inoculum and after the denitrification treatment was treated in the same manner as in example 1₃ ^--N), ammonia Nitrogen (NH)₄ ⁺N), total nitrogen (in TN), chemical oxygen demand COD were determined.

The results are shown in Table 2, and it is understood from the data shown that the main pollutant in the river water is nitrate nitrogen. After 3 days of microbial inoculum denitrification treatment in natural environment, the nitrate nitrogen content in the water body is reduced to 0.5mg/L from 12.5mg/L, and the nitrate nitrogen removal rate is over 90 percent. After the microbial inoculum repairs for three days, the Chemical Oxygen Demand (COD) of the water area of the section is slightly reduced.

TABLE 2 treatment Effect of aerobic denitrifying strains on urban rivers

Industrial applicability

The aerobic denitrifying strain pseudomonas N200 is suitable for removing nitrogen in biological treatment of industrial-grade reclaimed water, sewage and the like, and is also suitable for being directly thrown into a natural water body to reduce the nitrate nitrogen content, the total nitrogen content and the like in the water body with higher nitrate nitrogen content in the natural environment, so that the eutrophication of the water body is effectively relieved at extremely low cost, and the environments of rivers and lakes are restored.

When the pseudomonas N200 is put into a water body in a microbial inoculum form to eliminate the nitrate nitrogen content in the water body, the pseudomonas N200 has no toxicity to various organisms in the water body such as fishes and shrimps, and is a safe and friendly nitrogen remover, so that the pseudomonas N200 can be expected to be applied to the aspects of environmental protection and aquatic products.

Claims

1. Pseudomonas bacteria (Pseudomonas sp.)N200The preservation number is CGMCC No. 18072.

2. The pseudomonad of claim 1N200The 16S rDNA sequence is shown in SEQ ID No. 1.

3. The pseudomonad of claim 1 or 2N200It can utilize nitrate and ammonium salt to make denitrification.

4. A denitrification method characterized by using the Pseudomonas bacterium according to any one of claims 1 to 3N200Wherein the denitrification condition is as follows: the dissolved oxygen DO is 3-7mg/L, the temperature is 15-42 ℃, and the pH is 6-10.

5. The denitrification process according to claim 4, comprising the steps of:

a step of fungus feeding: adding a microbial inoculum into water with dissolved oxygen of 3-7mg/L according to the volume ratio of 1/100-1/1000 to the water, wherein the microbial inoculum comprises 1 x 10⁸CFU/mL or OD₆₀₀Pseudomonas of any of claims 1 to 3 having a value of 1N200；

An amplification step: placing the pseudomonas at room temperatureN200 1-5 days;

a purification step: and introducing water to be purified, wherein dissolved oxygen is kept at 3-7mg/L, and obtaining the water with reduced nitrate nitrogen content.

6. A microbial agent or water purifying agent comprising the Pseudomonas bacterium according to any one of claims 1 to 3N200Wherein the microbial agent or water purifying agent further comprises one or more selected from stabilizer, thallus preservative, nutritional agent, taste masking agent, osmotic pressure regulator, colorant, and pH indicatorAnd (4) seed preparation.

7. The microbial agent or water purifying agent according to claim 6, which is in the form of liquid culture or lyophilized powder.

8. Pseudomonas as claimed in any one of claims 1 to 3N200The use of the microbial inoculum or water purifying agent of claim 6 or 7 in denitrification for nitrogen-containing bodies of water.

9. Use according to claim 8, wherein the water body is selected from water bodies for aquaculture, domestic sewage or rivers and lakes.

10. The use according to claim 9, wherein the body of water for aquaculture comprises a body of water for direct aquaculture, and waste water discharged during or after aquaculture.