CN104164389A

CN104164389A - Pseudomonas fluorescens and application thereof in aquaculture

Info

Publication number: CN104164389A
Application number: CN201410335916.2A
Authority: CN
Inventors: 武玉强; 苟万里; 刘广
Original assignee: QINGDAO WEILAN TIANCHENG BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: Qingdao Weilan LongChu Biotechnology Co.,Ltd.
Priority date: 2014-07-09
Filing date: 2014-07-09
Publication date: 2014-11-26
Anticipated expiration: 2034-07-09
Also published as: CN104164389B

Abstract

The invention relates to the technical field of functional microbial screening, and in particular provides a Pseudomonas fluorescens HF-3 strain with the preservation number of CCTCC NO:M2014274. The strain has the aerobic denitrification property, and can efficiently degrade nitrite nitrogen in aquaculture sewage, 48h after the treatment, the removal rate of the nitrite nitrogen in the water reaches 94.61%, and the effect is remarkable; the Pseudomonas fluorescens HF-3 strain can be used for directly processing water in a shed shrimp aquaculture pool, and 48h after the treatment, about 94.35% of the nitrite nitrogen can be removed. The Pseudomonas fluorescens HF-3 strain can effectively degrade the nitrite nitrogen in the domestic wastewater, and the removal rate is higher than 90%, so that the Pseudomonas fluorescens HF-3 strain can be developed into aquaculture probiotics and sewage treatment agents, and has broad application prospects.

Description

One fluorescent pseudomonads and the application in aquaculture thereof

Technical field

The present invention relates to functional microorganism triage techniques field, be specifically related to Pseudomonas fluorescens and application thereof that a strain has aerobic denitrification function.

Background technology

In recent years, China's culture fishery fast development, intensive, high-density breeding scale expanding day, concentrated bait throwing in causes and in aquaculture water, accumulates a large amount of residual baits, ight soil and animals and plants corpse, cause water body nitrogen element content severe overweight, eutrophication aggravation, is destroyed breeding ecological environment, fish and shrimp diseases takes place frequently, and finally causes huge financial loss.Nitrifying process is by NO ₂ ^--N is converted into NO ₃ ^--N is the important ring in occurring in nature nitrogen cycle.Along with the highdensity development of aquaculture, daily ration, feeding quantity continues to rise, and occurring in nature denitrifying load continues to increase, and causes the accumulation of nitrogen.Research shows, NO ₂ ^--N is one of pathogenic main contributor of aquatic animal, NO ₂ ^--N can be preferentially combined with oxyphorase and hemocyanin, causes cultivation object anoxic, and then causes various diseases.So NO in control aquaculture water ₂ ^--N becomes the emphasis that aquaculture water quality is processed, and is also the difficult point of current water conditioning.

Because bio-denitrification technology has that cost is low, easy to operate, qualified discharge reliability is strong and the advantage such as non-secondary pollution, become gradually the major way that industrial sewage, sanitary sewage and aquiculture waste water are processed.Aerobic denitrification is that the bio-denitrification technology with unique advantage is compared in anoxic denitrification denitrogenation that propose in recent years and traditional: on the one hand, under aerobic conditions, carry out denitrification, nitrification and denitrification can be carried out simultaneously in a reactor, and equipment and manipulation cost declines to a great extent; On the other hand, the product of nitrification can avoid suppressing nitrification directly as the substrate of denitrification, the aggravation of nitrification and denitrification process.At present, aerobic denitrification more and more receives people's concern, also many for the screening study of aerobic denitrifying bacteria both at home and abroad, but its achievement still can not meet industrial actual demand far away.

Summary of the invention

The present invention, for solving prior art problem, provides a strain novel fluorescence pseudomonas and the application aspect water body purification thereof.The water sample of described Pseudomonas fluorescens screening in salmon circulating water cultivation waste-water treatment tower, has heterotrophism and Nitrification, can significantly reduce the NO in water body ₂-N content, can be widely used in the purifying treatment of aquaculture water and sanitary sewage.

One aspect of the present invention provides a fluorescent pseudomonads (Pseudomonas fluorescens) HF-3, on June 24th, 2014, be preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2014274.

The application of above-mentioned Pseudomonas fluorescens HF-3 bacterial strain in breeding wastewater or sanitary sewage disposal.

The present invention also provides a kind of microbe additive, is with preparing after Pseudomonas fluorescens HF-3 fermentation.

Above-mentioned microbe additive is bacterium powder.

Beneficial effect of the present invention:

The Pseudomonas fluorescens HF-3 bacterial strain that the present invention screens, has aerobic denitrification capability, and the nitrite nitrogen in energy efficient degradation breeding wastewater, processes after 48h, and in water body, the clearance of nitrite nitrogen reaches 94.61%, and effect is remarkable; Utilize described Pseudomonas fluorescens HF-3 bacterial strain directly to process the water body in hut shrimp aquaculture pond, process after 48h, can remove approximately 94.35% nitrite nitrogen.The nitrite nitrogen that Pseudomonas fluorescens HF-3 bacterial strain of the present invention can also effectively be degraded in sanitary sewage, clearance is higher than 90%.Therefore, this bacterial strain can be developed as aquatic products probiotics and sewage-treating agent, has broad application prospects.

Embodiment:

Embodiment 1: the separation of bacterial strain, screening and evaluation

1, sample

Take from the water sample in east, Yantai ocean group salmon circulating water cultivation waste-water treatment tower;

2, aerobic denitrification substratum:

Title	Consumption
		KNO ₃	1.0g
KH ₂PO ₄	1.0g
		Fecl ₂·6H ₂O	0.5g
CaCl ₂·2H ₂O	0.2g
		MgSO ₄·7H ₂O	1.0g
Sodium succinate	8.5g
		Dibromothymolsulfonphthalein (1% ethanolic soln)	1.0g
Agar	20.0g
		Pure water	Be settled to 1000mL

3, screening method

Adopt gradient dilution method that water sample is diluted to proper concn; The water sample of getting after 0.1mL dilution is evenly coated aerobic denitrification media surface, inserts constant incubator, cultivates after 2～3d at 30 ℃, and the single bacterium colony with on transfering loop picking substratum, carries out separation and purification, is primary dcreening operation bacterial strain.

This test altogether picking 10 strain list bacterium colonies, and called after HF-1, HF-2, HF-3 according to this ..., HF-10.Inoculate single bacterium colony to nutrient broth medium, under 30 ℃, 160r/min condition, carry out after liquid culture 24h, aseptic centrifugal, with the resuspended bacterium mud of physiological saline, make bacterium liquid, wherein bacteria concentration is 10 ⁸～10 ⁹cFU/mL.

In the ratio of 1% (v/v), above-mentioned bacterium liquid is inoculated into respectively to (NO in sterilized breeding wastewater ₂-N concentration is 1.3mg/L), under 30 ℃, 160r/min condition, carry out shaking flask processing, NO in sampling and measuring breeding wastewater respectively after 48h ₂the concentration of-N, calculates NO ₂the clearance of-N, concrete outcome is as shown in table 1:

NO ₂the concentration of-N adopts diazonium-azo light-intensity method to measure;

Clearance=(starting point concentration-termination concentration)/starting point concentration * 100%.

NO in water body after table 1 processing 48h ₂the removal situation of-N

Strain number	Initial NO ₂ ^--N(mg/L)	Stop NO ₂ ^--N(mg/L)	Clearance
				HF-1	1.30	0.84	35.38％
HF-2	1.30	0.58	55.38％
				HF-3	1.30	0.07	94.61％
HF-4	1.30	0.19	85.38％
				HF-5	1.30	0.44	66.15％
HF-6	1.30	0.92	29.23％
				HF-7	1.30	1.21	6.92％
HF-8	1.30	0.90	30.77％
				HF-9	1.30	0.71	45.38％
HF-10	1.30	0.45	65.38％

From the data of table 1, can find out, the bacterial strain HF-3 that the present invention filters out is to NO in breeding wastewater ₂the degradation effect of-N is best, NO after processing for 48 hours ₂the clearance of-N is the highest, is 94.61%, far above other bacterial strains that screen, therefore can be applicable to water body purification field.

4, identification of strains

1) colony morphology characteristic: above-mentioned bacterial strains HF-3 is cultivated 18 hours on nutrient agar, be rendered as white transparence, surface wettability, non-wrinkled, diameter 1mm bacterium colony, its cell of microscopy is rod-short.

2) utilize test kit to extract the genomic dna of above-mentioned bacterial strains HF-3, utilize round pcr amplification 16S rRNA sequence, after order-checking, obtain the fragment that length is 1408bp.By BLAST compare of analysis, it is 99% with the 16S rRNA sequence similarity degree of many pseudomonas of having announced, identifies and confirms that bacterial strain HF-3 is pseudomonas, consistent with biochemical identification result.The 16S rRNA sequence of bacterial strain HF-3:

AGTCGAGCGGTAGAGAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAAT

GCCTAGGAATCTGCCTAGTGGTGGGGGATAACGTTCGGAAACGGACGCTAATACCGC

ATACGTCCTACGGGAGAAAGCGGGGGACCTTCGGGCCTCGCGCCATTAGATGAGCCT

AGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTG

GTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGG

AGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGT

GTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGTAGTAAC

TTAATACGTTGCTACTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCA

GCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG

CGCGTAGGTGGTTCAGTAAGTTGGAAGTGAAATCCCCGGGCTCAACCTGGGAACTGC

TTTCAAAACTGCTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGG

TGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGAT

ACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT

CCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGTCTTGAACTCTTAGTGGCGCAG

CTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGA

ATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGA

AGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCTAGAGATAGATTGGTGCCTTCG

GGAGCTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG

TTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTAATGGTGGGA

ACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCA

TCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTG

CCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCT

GCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGT

GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAC

CAGAAGTAGCTAGTCTAACCGCAAGGGGGACGGTACCACGGTGTT

3) by above-mentioned HF-3 bacterial strain called after Pseudomonas fluorescens (Pseudomonas fluorescens) HF-3, and on June 24th, 2014, being preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University, its deposit number is CCTCC NO:M2014274.

Embodiment 2: Pseudomonas fluorescens HF-3 processes aquaculture system experiment

Pseudomonas fluorescens HF-3 is activated, and enlarged culturing, through liquid state fermentation, the dry approximately bacterium powder of 10,000,000,000/g of making of spraying.

Experiment is located at the hut shrimp aquaculture pond of Jiangsu Rudong to be carried out, and chooses three culturing pools that water-quality guideline is close parallel as three, with plastic cloth, the water body of culturing pool is separated respectively, and half water body is as experimental group, and second half water body as a control group.First distinguish NO in test experience group and control group water body ₂ ^-the starting point concentration of-N then adds the above-mentioned Pseudomonas fluorescens HF-3 of 10ppm bacterium powder in experimental group water body, and control group does not add.The uninterrupted aeration of experimental session, and it is consistent respectively to organize aeration rate.Experimental temperature adopts 28-30 ℃, measures respectively NO in each experimental group and control group water body after 48h ₂ ^-the concentration of-N, averages the detected result of three parallel group, calculates clearance.Concrete outcome is as shown in table 2:

NO in the water body of hut shrimp aquaculture pond after table 2 processing 48h ₂the removal situation of-N

Group	Initial NO ₂ ^--N(mg/L)	Stop NO ₂ ^--N(mg/L)	Clearance
				Control group	1.27±0.03	1.14±0.02	10.24％
Experimental group	1.24±0.03	0.07±0.02	94.35％

From above-mentioned experimental result, the Pseudomonas fluorescens HF-3 that the present invention screens can effectively remove the NO in water body ₂-N, NO in the water body of hut shrimp aquaculture pond after processing 48h ₂the clearance of-N reaches 94.35%, and the clearance of control group is only 10.24%, and effect is very obvious.Therefore, Pseudomonas fluorescens HF-3 of the present invention can be widely used in aquaculture, for solving the too high problem of aquaculture water Central Asia nitrate nitrogen content.

Nitrite nitrogen in energy degrading cultivation water, the nitrite nitrogen that Pseudomonas fluorescens HF-3 of the present invention can effectively degrade in sanitary sewage equally, NO after processing 48h ₂the clearance of-N is higher than 90%.

Claims

1. a Pseudomonas fluorescens, is characterized in that, the deposit number of described Pseudomonas fluorescens is CCTCC NO:M2014274.

2. the application of Pseudomonas fluorescens claimed in claim 1 in water body purification field.

3. a microbial preparation, described microbial preparation includes Pseudomonas fluorescens claimed in claim 1.

4. the application of microbial preparation claimed in claim 3 in breeding wastewater or purifying domestic sewage processing.