CN113174345A

CN113174345A - Heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof

Info

Publication number: CN113174345A
Application number: CN202110523474.4A
Authority: CN
Inventors: 高娜; 梁阳阳; 杨坤; 李静; 方婷; 赵秀侠; 卢文轩; 洪大保
Original assignee: Fisheries Research Institute of Anhui AAS
Current assignee: Fisheries Research Institute of Anhui AAS
Priority date: 2021-05-13
Filing date: 2021-05-13
Publication date: 2021-07-27

Abstract

The invention provides a heterotrophic nitrification-aerobic denitrification strain with high-efficiency denitrification, which is a Pseudomonas strain (Pseudomonas mendocina) xh7, is preserved in China center for type culture collection, and has a preservation number of CCTCC NO: m2021275. The strain realizes the synchronous removal of ammonia nitrogen, nitrite and nitrate nitrogen under aerobic conditions, and has high removal efficiency; and the nitrification and denitrification are carried out in a coupling way, so that the problems of slow proliferation of autotrophic nitrifying bacteria and long hydraulic retention time of the system can be effectively solved.

Description

Heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof

Technical Field

The invention belongs to the field of environmental microorganisms, and particularly relates to a heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof.

Background

In aquaculture, nitrogen pollution is one of the important factors causing the deterioration of aquaculture water quality, and seriously threatens the survival of aquatic organisms. The microorganisms have an important role in nitrogen conversion, and can convert harmful nitrogen pollutants in the water body into gaseous substances to be discharged out of the water body through ammoniation, nitrification and denitrification. Biological denitrification is of great interest because of its economy, high efficiency, green and environmental protection. Conventional biological denitrification generally involves two processes: the first step is nitrification, namely nitrifying bacteria react NH under aerobic conditions₄ ⁺-N via NO₂ ^-Conversion of-N to NO₃ ^--N; the second step is that denitrifying bacteria react NO under anaerobic conditions₃ ^--N、NO₂ ^--reduction of N to gaseous nitrogen. Since bacteria participating in these two processes have different degrees of tolerance to oxygen and organic matters, the conventional biological denitrification system comprises two separate systems, resulting in low denitrification efficiency and increased denitrification cost. With the progress of research, heterotrophic nitrifying bacteria are gradually discovered and separated, and can convert nitrogen-containing compounds in water into hydroxylamine and NO through nitrification while utilizing organic matters in water to carry out self growth₂ ^--N or NO₃ ^--N, etc.; meanwhile, most bacteria can simultaneously carry out aerobic denitrification to remove NO₂ ^--N or NO₃ ^-N is converted into a nitrogen-containing gas. In recent years, a plurality of microorganisms with heterotrophic nitrification and aerobic denitrification functions are separated from soil, deep-sea craters, sludge, lake water and the like in succession, and the microorganisms are a microbial resource with important application value. The discovery of the above components solves the problems of long starting time, poor stability, strict requirements on the conditions of the nitrification link, nitrification and denitrification of the traditional biological denitrification treatmentThe method has the defects of incapability of synchronous operation and the like, is expected to overcome the contradiction between the treatment efficiency and economic use of the traditional treatment process, and has good development prospect. However, in terms of the current research situation, the heterotrophic nitrification-aerobic denitrification bacteria have fewer species and poor wastewater treatment effect, so that the expansion of the heterotrophic nitrification-aerobic denitrification bacteria strain resources, the improvement of the wastewater treatment efficiency and the enhancement of the practicability are urgently needed.

Disclosure of Invention

In view of the above problems and disadvantages, the present invention aims to provide a bacterium having heterotrophic nitrification-aerobic denitrification activity; another purpose is to provide a culture method and a using method for screening the strain from the bottom mud of the aquaculture pond, so that the strain can not only rapidly remove ammonia nitrogen, but also can grow under the condition that nitrite or nitrate is used as a unique nitrogen source, and can effectively remove nitrogen in the nitrite or nitrate. The strain takes sodium citrate as a carbon source, aerobically degrades ammonia nitrogen, nitrate nitrogen and the like in a water body, hardly accumulates nitrite nitrogen in the denitrification process, and avoids secondary pollution.

A heterotrophic nitrification-aerobic denitrification strain with high denitrification efficiency is a Pseudomonas strain (Pseudomonas mendocina) xh7 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021275, preservation date: year 2021, month 3, day 25, address: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.

In a further embodiment, the effective sequence length of the 16SrDNA of the Pseudomonas strain (Pseudomonas mendocina) xh7 is 1500bp, and the sequence is shown as SEQ ID NO: 1 is shown.

In a further scheme, the Pseudomonas strain (Pseudomonas mendocina) xh7 is obtained by enrichment culture, separation and purification of culture pond sediment.

In a further embodiment, the Pseudomonas strain (Pseudomonas mendocina) xh7 is grown with ammonia nitrogen, nitrate or nitrite as the sole nitrogen source.

Another object of the present invention is to provide the use of the above heterotrophic nitrification-aerobic denitrification bacterial strain for efficient denitrification, wherein the Pseudomonas mendocina (Xh 7) is used for denitrification of nitrogen-containing water.

In a further scheme, the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen, nitrite nitrogen or nitrate nitrogen.

In a further scheme, the Pseudomonas strain (Pseudomonas mendocina) xh7 is cultured by heterotrophic nitrification and enrichment from bottom sludge of an aquaculture pond, then cultured by an aerobic denitrification screening medium, a chromogenic culture medium and an R2A liquid culture medium in sequence, and then inoculated in a nitrogen-containing water body for aerobic continuous culture.

In a further scheme, the aerobic continuous culture utilizes sodium citrate as a carbon source, and the temperature is 30-35 ℃; the inoculation amount of the Pseudomonas strain (Pseudomonas mendocina) xh7 is 1 percent of the volume of the nitrogen-containing water body.

The invention provides application of the bacteria in biological denitrification of water, and the bacteria provided by the invention can effectively remove ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in water.

Compared with the prior art, the Pseudomonas sp.xh7 strain and the application thereof provided by the invention have the following beneficial effects:

(1) the Pseudomonas sp.xh7 provided by the invention can grow by respectively using ammonia nitrogen, nitrite and nitrate as unique nitrogen sources, can synchronously remove the ammonia nitrogen, nitrite and nitrate nitrogen under aerobic conditions, has high removal efficiency, solves the bottleneck problem that aerobic nitrification and anoxic denitrification sectional treatment are required in biological denitrification in the traditional wastewater treatment, is coupled with nitrification and denitrification, can well compensate acidity generated in the nitrification process by alkalinity generated in the denitrification process, does not need to add alkali to adjust pH in the whole process, has high growth rate and high cell yield compared with autotrophic nitrification, and can effectively solve the problems of slow proliferation of autotrophic nitrifying bacteria and long hydraulic retention time of a system;

(2) the strain can complete synchronous removal of carbon and nitrogen organisms without constructing a new reactor, simplifies the process flow to the maximum extent, saves the cost of equipment and investment, and has better economic benefit and environmental protection benefit.

Drawings

FIG. 1: the strain xh7 phylogenetically evolved tree, and shows that the strain with the closest relationship with the strain is Pseudomonas mendocina NBRC 14162;

FIG. 2: the growth characteristic of the strain xh7 with ammonia nitrogen as a unique nitrogen source;

FIG. 3: the strain xh7 has denitrification performance taking ammonia nitrogen as a unique nitrogen source, wherein a shows that the accumulation amount of nitrate nitrogen and nitrite nitrogen is less in the ammonia nitrogen removal process, and b shows that the total nitrogen removal rate reaches 100% in 32 hours;

FIG. 4: growth curves of strain xh7 using nitrate and nitrite as nitrogen sources.

FIG. 5: the denitrification property of the strain xh7 when nitrate and nitrite are used as nitrogen sources;

FIG. 6: NH removal by strain xh7 and strain WZUF22₄ ⁺Comparison of the N wastewater capacities.

Detailed Description

The present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

The media formulations used in the examples are as follows:

heterotrophic nitrification enrichment culture medium: (NH)₄)₂SO₄0.472g, 4.052g sodium succinate and 50ml Vickers salt solution, adding water for dissolution, supplementing distilled water to 1L, and adjusting the pH to 7.0. Wherein Vickers salt solution (g/L): k₂HPO₄ 5.0g，FeSO₄·7H₂O 0.05g，NaCl 2.5g，MgSO₄·7H₂O 2.5g，MnSO₄·4H₂O 0.05g。

Aerobic denitrification enrichment medium: 0.36g/L KNO₃，10.55g/L Na₂HPO₄·12H₂O，1.5g/LKH₂PO₄，0.1g/L MgSO₄·7H₂O, 4.0g/L sodium citrate and 0.2 percent (volume ratio) of trace element solution. Trace elementsSolution: 50g/L EDTA-Na2, 2.2g/L ZnSO₄，5.5g/L CaCl₂，5.06g/L MnCl₂·4H₂O，5.0g/L FeSO₄·7H₂O，1.57g/L CuSO₄·5H₂O，1.61g/L CoCl₂·6H₂O。

GN chromogenic Medium: 1.0g/L KNO₃8.5g/L sodium citrate, 1.0g/L L-asparagine, 1.0g/LKH₂PO4，1.0g/L MgSO₄·7H₂O，0.2g/L CaCl₂·6H₂O，0.05g/L FeCl₃·6H₂O, 0.1% by volume of 1% by mass of bromothymol blue (BTB), and the pH was adjusted to 7.0. The liquid chromogenic medium was added with 2% agar to prepare a solid chromogenic medium. Sterilizing at 121 deg.C for 20 min.

R2A medium: 0.5g/L of tryptone, 0.5g/L of yeast extract, 0.5g/L of starch, 0.5g/L of enzymatic hydrolysis casein, 0.5g/L of glucose, 0.3g/L of sodium pyruvate, 0.3g/L of dipotassium hydrogen phosphate and 0.024g/L of magnesium sulfate.

Heterotrophic nitrification determination of culture medium NM (NH)₄)₂SO₄0.945g/L, 16.34g/L sodium citrate and MgSO₄·7H₂O 1g/L，KH₂PO₄ 0.25g/L，Na₂HPO₄ 0.3g/L。

Aerobic denitrification determination culture medium DMA: KNO₃0.722g/L, sodium citrate 5g/L, MgSO₄·7H₂O1g/L，KH₂PO₄ 0.25g/L，Na₂HPO₄ 0.3g/L。

Aerobic denitrification assay medium DMB: NaNO₂0.5g/L, 5g/L sodium citrate, MgSO₄·7H₂O 1g/L，KH₂PO₄ 0.25g/L，Na₂HPO₄ 0.3g/L。

Example 1: the screening of heterotrophic nitrification-aerobic denitrification strains comprises the following specific steps:

1. isolation and purification of bacteria

Inoculating the bottom mud collected from the culture pond into a heterotrophic nitrification enrichment medium according to the proportion of 10 percent of the volume ratio, and carrying out shaking table culture at the temperature of 30 ℃ and at the speed of 150 r/min; the culture was continued for 20 days by replacing the fresh heterotrophic nitrification-enrichment medium at a volume ratio of 10% every 48 hours.

Inoculating the domesticated microorganisms into an aerobic denitrification enrichment medium for further screening, wherein the aerobic denitrification enrichment medium takes potassium nitrate as a unique nitrogen source.

1mL of sample subjected to primary screening by the aerobic denitrification enrichment medium is uniformly coated on a solid GN chromogenic medium and then is cultured in a constant temperature incubator at 30 ℃.

To further verify that the single colony picked has the property of turning GN color medium blue, the single colony of blue color in solid GN color medium was picked and inoculated into 3mL of sterilized liquid GN color medium, shake-cultured at 30 ℃ and 150r/min, the color change was observed, and a strain capable of turning GN medium from green to blue was selected as a rescreen.

2. Molecular biological identification

Single colonies of the rescreened bacteria were picked for 16S rRNA gene amplification and PCR amplification was performed using bacterial 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3').

Wherein the 50 mu L PCR reaction system comprises: 2 XTaq PCR Master Mix 25. mu.L, 27F (10. mu.M) 1. mu.L, 1492R (10. mu.M) 1. mu.L, DNA template as single colony picked, ddH₂O24. mu.L. Setting a PCR program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 40s, and amplification for 30 cycles; final extension at 72 ℃ for 10 min. And recovering and purifying PCR products by using a gel recovery kit, cloning and transforming, screening a positive clone sub-colony, sequencing after amplification culture, submitting a 16S rDNA sequence of the bacteria obtained by sequencing to an EZBioCloud database (https:// www.ezbiocloud.net/identity) for sequence comparison, and determining the species most similar to the target strain according to the highest sequence integrity (completensiss) and Similarity (Similarity).

The effective sequence length of the sequenced 16S rDNA is 1500bp, and the sequence is shown as SEQ ID NO: 1 is shown. The genetic relationship between the strain and other strains is shown in figure 1, and the 16SrDNA sequence of the bacterium is confirmed to have homology of more than 99 percent with Pseudomonas mendocina (Pseudomonas mendocina) published in a database through Blastn analysis and phylogenetic evolution tree construction, so that the bacterium is confirmed to be a Pseudomonas and named as Pseudomonas xh 7.

The pseudomonas xh7 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan university school with No. 299 in Wuhan district, Wuhan city, Hubei province, and the preservation number is CCTCC NO: m2021275.

Example 2: determination of ammonia nitrogen degradation capability of strain

The rescreened bacterial colonies screened in example 1 were picked up and cultured in R2A liquid medium at 30 ℃ for 18 hours at 200rpm in a gas bath shaker, centrifuged at 4000 rpm for 2 minutes, the cells were collected, resuspended in equal amounts of sterile water, and inoculated in NM (heterotrophic nitrification enrichment medium) liquid medium at 1% (volume ratio) inoculum size. Shaking-flask culture at 30 deg.C and 200rpm, and periodically sampling to determine optical density (OD600), Total Nitrogen (TN), and ammonia Nitrogen (NH)₄ ⁺-N), Nitrate (NO)₃ ^--N) and Nitrite (NO)₂ ^--N) concentration, and a growth curve of the cells was prepared from the OD600 value. As shown in FIGS. 2 and 3, the strain grows rapidly, namely the strain enters a logarithmic phase growth after 8 hours, and enters a plateau phase after 48 hours, and the maximum OD600 value reaches 2.173. At the moment, the removal rate of the strains on the total nitrogen reaches 99.7 percent, the accumulation amount of nitrite nitrogen is very little, and no secondary pollution is generated.

Example 3: determination of aerobic denitrification capability of strain

The rescreened bacterial colonies screened in example 1 were picked up to R2A liquid medium, cultured for 18 hours at 30 ℃ in a gas bath shaker at 200rpm, centrifuged at 4000 rpm for 2min, collected, resuspended in equal amount of sterile water, and inoculated into DMA and DMB liquid media at 1% (volume ratio) inoculum size, respectively. Shaking-flask culture at 30 deg.C and 200rpm, and periodically sampling to determine optical density (OD600), Total Nitrogen (TN), and ammonia Nitrogen (NH)₄ ⁺-N), Nitrate (NO)₃ ^--N) and Nitrite (NO)₂ ^--N) concentration, and a growth curve of the cells was prepared from the OD600 value. In the graph 5, when a uses nitrate as the only nitrogen source, the total nitrogen removal rate is 92.8 percent after the strain is cultured for 24 hours; in FIG. 5 b is nitrous acidWhen the salt is the only nitrogen source, the total nitrogen removal rate of the strain after being cultured for 32 hours reaches 91.9 percent. As can be seen from FIGS. 4 and 5, the strain xh7 takes nitrate as a unique nitrogen source, and enters a plateau stage after being cultured for 16 hours, wherein the removal rate of nitrate nitrogen is 100%, and the removal rate of total nitrogen is the highest and reaches 92.8% after being cultured for 24 hours. When the bacterial strain takes nitrite as a unique nitrogen source, the bacterial strain is cultured for 24 hours and grows in a plateau stage, the removal rate of nitrite nitrogen is 95.2%, and the removal rate of total nitrogen is the highest and reaches 91.9% after culturing for 32 hours. As can be seen from the above, the strain xh7 has obvious aerobic denitrification capability.

Example 4: strain removal of artificially prepared NH₄ ⁺-N Sewage Process

The strain is compared with another strain of pseudomonas mendocina strain WZUF22 (with the preservation number of CGMCCNo.7523 strain, and the patent application publication number CN 103484398A) under the same experimental conditions. The experimental conditions were as follows: preserving the strain (2.0ml of frozen tube-thawed bacterial liquid), respectively inoculating the rescreened strain and WZUF22 strain screened in example 1 into a 500ml conical flask filled with 200ml of LB culture medium, culturing at 30 ℃ and 150rpm for 24h, centrifuging at 8000rpm for 10min to obtain thallus, washing with sterile water for 2 times to prepare bacterial suspension with OD680 of 0.900-1.000; then respectively transferring the mixture to 1g/L NH prepared by manpower according to the inoculation amount of 5 percent (V/V)₄Cl (methanol) in 200ml of 500ml conical flask culture medium, formula: NH₄Cl 1g, sodium citrate 10g, MgSO₄·7H₂O 0.1g，K₂HPO₄ 0.5g，NaCl 0.2g，MnSO₄·4H₂O 0.02g，FeSO₄ 0.02g，H₂O1000 ml, pH7) at 30 ℃ and 150rpm (DO value 4.3 mg. multidot.L)^-1) The culture was incubated at low temperature, biomass (OD680) was measured by time sampling, and NH of the supernatant was measured after centrifugation at 8000rpm for 10min₄ ⁺Concentration of-N, NO₂ ^--N concentration and NO₃ ^--N concentration, calculating NH₄ ⁺Removal rate of-N and NO₃ ^--N、NO₂ ^--accumulation of N.

As shown in a and b in FIG. 6, the strain xh7 will react with 1g/L NH within 24h₄NH contained in Cl₄ ⁺The N removal is 85.95 percent, which is improved compared with the strain WZUF2212.45 percent. Strain xh7 at NH₄ ⁺The growth performance in N sewage is better than that of the strain WZUF22, and the OD680 reaches 2.151 in 24 hours. In addition, strain xh7 accumulated significantly less NO 3-N and NO 2-N during NH4+ -N removal compared to strain wzaf 22; and nitrite is always at a lower level, with less harm.

In conclusion, the strain xh7 has obvious advantages in removing NH4+ -N sewage compared with the strain WZUF 22.

The above-described embodiments are merely preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above-described embodiments, and it should be understood that many other modifications and embodiments can be devised by those skilled in the art, which will fall within the spirit and scope of the principles of this disclosure.

Sequence listing

<110> aquatic research institute of agricultural science institute of Anhui province

<120> heterotrophic nitrification-aerobic denitrification strain for efficient denitrification and application thereof

<141> 2021-04-16

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1500

<212> DNA

<213> Pseudomonas mendocina strain xh7

<400> 1

agagtttgat cctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60

ggatgaagag agcttgctct ctgatttagc ggcggacggg tgagtaatgc ctaggaatct 120

gcctggtagt gggggataac gttccgaaag gaacgctaat accgcgtacg tcctacggga 180

gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt 240

ggtgaggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300

cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360

atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420

cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac gttaccgaca 480

gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta 540

atcggaatta ctgggcgtaa agcgcgcgta ggtggttcgt taagttggat gtgaaagccc 600

cgggctcaac ctgggaactg catccaaaac tggcgagcta gagtacggta gagggtggtg 660

gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720

accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780

accctggtag tccacgccgt aaacgatgtc aactagccgt tggaatcctt gagattttag 840

tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 900

aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960

aagaacctta cctggccttg acatgctgag aactttccag agatggattg gtgccttcgg 1020

gaactcagac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080

gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cctcgggtgg gcactctaag 1140

gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta 1200

cggccagggc tacacacgtg ctacaatggt cggtacaaag ggttgccaag ccgcgaggtg 1260

gagctaatcc cataaaaccg atcgtagtcc ggatcgcagt ctgcaactcg actgcgtgaa 1320

gtcggaatcg ctagtaatcg tgaatcagaa tgtcacggtg aatacgttcc cgggccttgt 1380

acacaccgcc cgtcacacca tgggagtggg ttgctccaga agtagctagt ctaaccttcg 1440

gggggacggt taccacggag tgattcatga ctggggtgaa gtcgtaacaa ggtagccgta 1500

Claims

1. A heterotrophic nitrification-aerobic denitrification strain for efficient denitrification is characterized in that: the strain is Pseudomonas strain (Pseudomonas mendocina) xh7 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021275.

2. The heterotrophic nitrification-aerobic denitrification strain with high denitrification efficiency as claimed in claim 1, wherein: the effective sequence length of the 16SrDNA of the Pseudomonas strain (Pseudomonas mendocina) xh7 is 1500bp, and the sequence is shown as SEQ ID NO: 1 is shown.

3. The heterotrophic nitrification-aerobic denitrification strain with high denitrification efficiency as claimed in claim 1, wherein: the Pseudomonas strain (Pseudomonas mendocina) xh7 is obtained by enrichment culture, separation and purification of bottom mud of an aquaculture pond.

4. The heterotrophic nitrification-aerobic denitrification strain with high denitrification efficiency as claimed in claim 1, wherein: the Pseudomonas strain (Pseudomonas mendocina) xh7 is grown with ammonia nitrogen, nitrate or nitrite as the sole nitrogen source.

5. The use of the highly denitrifying heterotrophic nitrification-aerobic denitrification strain according to any one of claims 1 to 4, wherein: the Pseudomonas strain (Pseudomonas mendocina) xh7 is used for nitrogen removal of nitrogen-containing water body.

6. Use according to claim 5, characterized in that: the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen or nitrate nitrogen.

7. Use according to claim 5, characterized in that: the Pseudomonas strain (Pseudomonas mendocina) xh7 is obtained by carrying out heterotrophic nitrification enrichment culture on bottom mud of an aquaculture pond, sequentially carrying out aerobic denitrification screening, chromogenic culture medium and R2A liquid culture medium culture, and then inoculating the culture medium into a nitrogen-containing water body for aerobic continuous culture.

8. Use according to claim 7, characterized in that: the aerobic continuous culture utilizes sodium citrate as a carbon source, and the temperature is 30-35 ℃; the inoculation amount of the Pseudomonas strain (Pseudomonas mendocina) xh7 is 1 percent of the volume of the nitrogen-containing water body.