CN102911897A - Cultural method for paracoccus denitrificans and application of same to purifying aquaculture water - Google Patents
Cultural method for paracoccus denitrificans and application of same to purifying aquaculture water Download PDFInfo
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- CN102911897A CN102911897A CN2012103831116A CN201210383111A CN102911897A CN 102911897 A CN102911897 A CN 102911897A CN 2012103831116 A CN2012103831116 A CN 2012103831116A CN 201210383111 A CN201210383111 A CN 201210383111A CN 102911897 A CN102911897 A CN 102911897A
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Abstract
The invention relates to a cultural method for paracoccus denitrificans and application of the same to purifying aquaculture water, and belongs to the technical field of aquaculture. Various low-value materials such as molasses and waste liquid of monosodium glutamate and production waste which are harmless for aquaculture organisms and the paracoccus denitrificans are used as raw materials, the raw materials are reasonably compounded into liquid culture media, paracoccus denitrifican liquid with high activity and high concentration is produced, the liquid is directly fed into the aquaculture water, an ecological environment of the aquaculture water is repaired, and an excellent water environment beneficial to survival and growth is created for the aquaculture organisms. The culture method and the application have the advantages that the paracoccus denitrificans become effective fast after being used, an inorganic nitrogen removal form and the removal speed can be selected according to an oxygen dissolving condition of the water, contents of harmful substances such as ammonia nitrogen, nitrite and nitrate in the water are remarkably reduced, the quality of the water environment and a survival condition for the aquaculture organisms are improved, the yield of the aquaculture organisms is increased, and the quality of the aquaculture organisms is improved.
Description
Technical field
The present invention relates to cultural method and the application in cultivation water purifies thereof of a kind of Paracoccus denitrificans, belong to technical field of aquaculture.
Background technology
In recent years, the development of the culture fishery of China is rapid, and the cultivation scale constantly enlarges.People are highly efficient and productive in order to pursue, and often drop into a large amount of bait and fertilizer that are rich in the nutritive substance such as nitrogen in aquaculture water, cause inorganic nitrogen (NH in the aquaculture water
4 +-N, NO
2 --N, NO
3 --N) excessive accumulation affects the water ecological setting quality.Nitrite nitrogen in the inorganic nitrogen is carcinogenic substance, can cause cell and tissue canceration, and can the interior ferrohemoglobin (Fe of the lively object of oxidizing water
2+) generation methemoglobin (Fe
3+), make it lose the ability of carrying oxygen molecule, thereby affect the transportation of oxygen in the hydrocoles body, make erythrodegeneration, cause biological death; The excessive meeting of nitric nitrogen makes the nutritive equilibrium imbalance in the water body, and quality of water environment reduces, and can change into nitrite nitrogen under local anaerobic condition.And ammonia nitrogen can generate nitrite nitrogen and nitric nitrogen by nitrosification and nitrification under aerobic conditions.Therefore for aquaculture water, in order to guarantee the quality and safety of aquaculture organism, must with inorganic nitrogen particularly the content of nitrite nitrogen and nitric nitrogen be controlled in the rational scope.
The conversion of nitrate nitrogen in water body and nitrite nitrogen and to remove mainly be that assimilation absorption and denitrification by microorganism realizes.It is generally acknowledged that denitrifying bacterium is the heterotrophism facultative anaerobic bacteria, only under anoxia condition just take nitrate or nitrite as electron acceptor(EA), carry out denitrification.Along with going deep into of research, the discovery of aerobic denitrifying bacteria shows under aerobic conditions and also can carry out denitrification.It is a Gospel that the discovery of aerobic denitrifying bacteria is controlled the environment of aquaculture water, because aquaculture water needs sufficient dissolved oxygen to guarantee, and realizes that under aerobic conditions denitrogenation provides a good approach for the nitrogen control of aquaculture water undoubtedly.In recent years, some researchs have been carried out in the aspects such as separation, evaluation, cultivation and application that Chinese scholars begins at aerobic denitrifying bacteria, have confirmed that Paracoccus denitrificans can carry out denitrification under aerobic condition.Paracoccus denitrificans is again the inorganic autotrophic bacteria of a kind of facultativeization energy simultaneously, can be at H
2, O
2, CO
2Carry out autophyting growth in the simultaneous situation, also can utilize the organism such as various carbohydrates, amino acid to carry out heterotrophic growth, therefore, it is at the lower extraordinary a kind of bacterial strain of realizing the nitrogen transformation effect of different condition (aerobic or anaerobism, autotrophy or heterotrophism).
The quality of aquaculture water environment has determined the eco-environmental quality of aquaculture water.Three nitrogen of excessive concentration (ammonia nitrogen, nitrite nitrogen, nitric nitrogen) are huge to aquatic animal and environmental hazard in the aquaculture water.Present aquaculture water denitrogenation modifying agent mainly is divided into chemistry and biological two large classes.The effect of chemical classes modifying agent is quick, and effect is directly perceived, and consumption is less, but effective object is single, and the time length is short, and often has ecological insecurity, cures the symptoms, not the disease; Biological species improving of environment agent is mainly take microbial preparations such as genus bacillus, EM bacterium as main, and is safe and effective, but onset time is slow, large usage quantity, and effect is subjected to environmental influence and unstable.Therefore the nitrate pollution control of cultivation water environment be badly in need of exploitation a kind of produce simple, cost is low, have the novel microorganism class improving of environment agent that denitrification effect is not subjected to the water body environment condition influence, to satisfy the needs of industry development.
Summary of the invention
The object of the invention is to overcome deficiency of the prior art, cultivation and the application method in cultivation water purifies of a kind of Paracoccus denitrificans are provided, utilize molasses, monosodium glutamate waste liquid etc. as substratum, cultivate the bacterium liquid of highly active Paracoccus denitrificans through liquid submerged fermentation.Bacterium liquid directly drops into aquaculture water, under different dissolved oxygen conditions, can remove targetedly or transform ammonia nitrogen, nitrite nitrogen or nitric nitrogen, the inorganic nitrogen composition that in the control water body environment aquaculture organism is harmful to, repair fast the aquaculture water ecotope, for aquaculture organisms is built a good water body environment.
According to technical scheme provided by the invention, the cultural method of a kind of Paracoccus denitrificans, culturing step is as follows: get Paracoccus denitrificans
Paracoccus denitrificans, ACCC10489 is from Chinese agriculture microbial strains preservation administrative center; Be inoculated in the LB liquid nutrient medium of 20 ~ 100mL sterilization, in 28 ~ 35 ℃, 80 ~ 150 rmin
-1Cultivate 20 ~ 30h on the shaking table; Then with in the LB liquid nutrient medium of its access 3 ~ 50L through sterilizing, in 28 ~ 35 ℃, 80 ~ 150 rmin
-1Cultivate 20 ~ 30h on the shaking table, it is for subsequent use to make the bacterial classification seed liquor;
The liquid fermentation medium that in 0.5 ~ 1.0 ton of stainless steel fermentor tank, adds bacterial classification seed liquor cumulative volume 65% ~ 75%, cooling is for subsequent use behind 115 ~ 121 ℃ of sterilization 15 ~ 30min; With above-mentioned bacterial classification seed liquor access fermentor tank, be to cultivate 24 ~ 30h under the condition of 20% ~ 30% saturated content at 28 ~ 35 ℃, dissolved oxygen content, treat bacterial concentration 〉=10
9CFU mL
-1The time with the phosphoric acid adjust pH be put into after 4 ~ 5 through 1 ~ 25L bucket of sterilization for subsequent use.
Described LB liquid nutrient medium is basic enrichment medium, contains peptone 5 ~ 15 gL
-1, yeast extract paste 3 ~ 8 gL
-1, NaCl 5 ~ 15 gL
-1, regulate pH 6.5-7.5, cooling is for subsequent use behind 115 ~ 121 ℃ of sterilization 15 ~ 30min.
Described liquid fermentation medium, its component ratio is counted by weight: 8 ~ 15 parts in the molasses of sugar content 〉=50%, 4 ~ 10 parts of monosodium glutamate waste liquids, MgSO
47H
20.01 ~ 0.1 part of O, K
2HPO
41.0 ~ 1.5 parts, KH
2PO
40.2 ~ 0.8 part, 78 ~ 88 parts in water;
In dissolving mixing in fermentor tank under the room temperature, the ratio of regulating molasses and monosodium glutamate waste liquid, so that solution C/N mol ratio reaches 10 ~ 15 ︰ 1, and with NaOH or HCl regulator solution pH value to 7.2～7.5,115 ~ 121 ℃ of sterilizations, for subsequent use after the cooling.
Described monosodium glutamate waste liquid COD is 10000 ~ 100000 mgL
-1, TN is 5000 ~ 20000 mgL
-1
The OD of described bacterial classification seed liquor
600Value is 0.8 ~ 1.2, and viable count is 1 * 10
8~ 9 * 10
10CFU mL
-1
The application method of a kind of Paracoccus denitrificans in cultivation water purifies adopts following steps: be 1 * 10 with bacteria concentration
8~ 9 * 10
10CFU mL
-1Paracoccus denitrificans bacterium liquid is by 500 ~ 1500 mL mu Mi Shuishen
-1Consumption o'clock join in the aquaculture water to be evolved in the morning 9 ~ 11; According to practical situation, used once every 5 ~ 10 days.
Result of use is relevant with the water body dissolved oxygen with inorganic nitrogen removal order.Water body dissolved oxygen 〉=6.0mgL
-1, 7 ~ 8 hours begin to work after using, and ammonia nitrogen begins to descend at first, and then nitrite nitrogen and nitric nitrogen begin to descend.Use rear 12 hours ammonia-nitrogen contents to reduce more than 70%, nitrate nitrogen content reduced more than 70% in 16 hours, and the nitrite nitrogen content is more than 70% after 22 hours; 3.0 mgL
-1≤ water body dissolved oxygen≤6.0 mgL
-1The time, 10 ~ 12 hours begin to work after using, and nitric nitrogen begins to descend at first, and then nitrite nitrogen and ammonia nitrogen begin to descend.Use rear 20 hours nitrate nitrogen contents to reduce more than 70%, the nitrite nitrogen content is more than 70% after 40 hours, and ammonia-nitrogen content reduced more than 70% in 60 hours; 0 mgL
-1≤ water body dissolved oxygen≤3.0 mgL
-1The time, using to begin to work in rear 24 hours, nitric nitrogen and nitrite nitrogen begin to descend simultaneously, and then ammonia nitrogen falls.Use rear 60 hours nitric nitrogens and nitrite nitrogen content more than 70%, ammonia-nitrogen content reduces more than 70% after 120 hours.
The present invention has following advantage: the present invention adopts aquaculture organisms and the harmless molasses of Paracoccus denitrificans, the multiple low value material such as monosodium glutamate waste liquid and production waste are raw material, through rationally being re-dubbed liquid nutrient medium and adopting the method for liquid submerged fermentation to produce the Paracoccus denitrificans bacterium liquid of high reactivity high density, and bacterium liquid directly dropped into aquaculture water, the ammonia nitrogen in the fast reducing cultivation water environment optionally in an orderly manner under different dissolved oxygen conditions, the pollution of the inorganic nitrogen such as nitrite nitrogen and nitric nitrogen, repair the aquaculture water ecotope, be conducive to the good water body environment of surviving and growing for one of aquaculture organisms construction.The present invention is with the main component of monosodium glutamate waste liquid as liquid nutrient medium, and cost is low, and is easy to use, realized sewage recycling, reduced pollution; The Paracoccus denitrificans concentration of producing is high, and is active strong, easy to use, can directly drop into aquaculture water, and usage quantity is little, and the consumption of every meter depth of water of every mu of water surface only is 500 ~ 1500mL; Instant effect and effect stability after using, can select the inorganic nitrogen form of required removal and remove speed according to water body dissolved oxygen situation, significantly reduce the content of the objectionable impuritiess such as ammonia nitrogen, nitrite, nitrate in the water body, reduce algae density, improve the environmental quality of water quality and substrate, improve the aquaculture organism life condition, effectively prevent aquaculture organism disease and sudden death, improve output and the quality of aquaculture organism.
Biological material specimens: employed bacterial strain Paracoccus denitrificans, see agriculture microbial strains in the Chinese microorganism strain composite catalog, its deposit number is 10489, and is open, takes from Chinese agriculture microbial strains preservation administrative center, sees the examination as to substances bibliography.
Embodiment
Following the present invention is further described in connection with embodiment: its described stainless steel fermentor tank is conventional equipment, buys in market, without any particular requirement, adopts common working method.
The cultivation of a kind of Paracoccus denitrificans of embodiment 1 the present invention and the application method in cultivation water purifies, adopt following processing step:
(1) cultivation of Paracoccus denitrificans:
Get from the test tube slant ring Paracoccus denitrificans (
Paracoccus denitrificans, ACCC10489, administrative center provides by the preservation of Chinese agriculture microbial strains) lawn, be inoculated in the LB liquid nutrient medium of 50mL sterilization, in 30 ℃, 100 rmin
-1Cultivate 24h on the shaking table.Then with in the LB liquid nutrient medium of its access 5L through sterilizing, in 30 ℃, 100 rmin
-1Cultivate 24h on the shaking table, it is for subsequent use to make the bacterial classification seed liquor.This moment bacterium liquid OD
600Value approximates 1.0, viable count 〉=10
9CFU mL
-1
The liquid fermentation medium that adds cumulative volume 350L in 0.5 ton of stainless steel fermentor tank, cooling is for subsequent use behind 121 ℃ of sterilization 20min.With above-mentioned bacterial classification seed liquor access fermentor tank, be to cultivate 24 hours under the condition of 20% saturated content at 30 ℃, dissolved oxygen content, treat bacterial concentration 〉=10
9CFU mL
-1The time with the phosphoric acid adjust pH be put into after 4.5 through the 25L bucket of sterilization for subsequent use.
Described LB liquid nutrient medium is basic enrichment medium, contains peptone 10 gL
-1, yeast extract paste 5 gL
-1, NaCl 10 gL
-1, regulate pH about 7.0, cooling is for subsequent use behind 121 ℃ of sterilization 20min.
Described liquid fermentation medium, its component ratio is counted by weight: 12 parts in molasses (sugar content 〉=50%), monosodium glutamate waste liquid (COD 〉=80000mgL
-1, TN 〉=16000mgL
-1) 8 parts, MgSO
47H
20.01 part of O, K
2HPO
41.0 part, KH
2PO
40.5 part, 84.49 parts in water, under the room temperature in fermentor tank dissolving mix, regulate the ratio of molasses and monosodium glutamate waste liquid so that solution C/N mol ratio reaches 10, and with NaOH or HCl regulator solution pH value to 7.2,121 ℃ of sterilizations, cooling is rear for subsequent use.
The action principle of each component is as follows among the present invention:
1) Paracoccus denitrificans: Paracoccus denitrificans (
Paracoccus denitrificans) belong to Proteobacteria (
Proteobacteria), first type distortion Gammaproteobacteria (
Alphaproteobacteria), red bacterium order (
Rhodobacterales), red bacterium section (
Rhodobacteraceae), paracoccus (
Paracoccus), be a kind of Gram-negative bacteria, spherical or rod-short cluster does not move.The about 2-3mm of colony diameter size, circular, complete, level and smooth, the White-opalescent in edge.It can be 5 ~ 37 ℃ of temperature, growth in pH7.0 ~ 8.5 scopes, and optimum growth temperature is 25 ~ 35 ℃, optimal pH is about 7.5.They live in aerobic or anaerobic environment in, strong adaptability is common in soil and nature or the artificial lake or ocean.Paracoccus denitrificans can carry out denitrification under aerobic condition, be again simultaneously that a kind of facultativeization can inorganic autotrophic bacteria.Can be at H
2, O
2, CO
2Carry out autophyting growth in the simultaneous situation, also can utilize the organism such as various carbohydrates, amino acid to carry out heterotrophic growth.In addition organic pollutant had very high degradation capability.The present invention mainly produces bacterium liquid with it as bacterial classification, utilizes its heterotrophic nitrification that has and aerobic denitrifying capacity, is implemented in the inorganic nitrogen of effectively administering aquaculture system under the dissolved oxygen variation condition greatly and pollutes.
2) molasses: molasses are byproducts of sugar industry, be a kind of thickness, chocolate, be semifluid object, composition is variant because of the difference of sugaring raw material, processing conditions, wherein mainly contain a large amount of fermentable sugars (mainly being sucrose), thereby be good fermentation raw material, can be used as substrate or the base-material of the fermented products such as yeast, monosodium glutamate, organic acid, can be used as raw material and the animal-feed of some food.Molasses output is larger beet sirup, cane molasses, grape molasses, corn molasses, and output is less the molasses of conversion and refinery molasses.Molasses contain a small amount of crude protein, be generally 3% ~ 6%, belong to the non-protein nitrogen(NPN) class more, such as ammonia, acid amides and nitrate etc., and amino-acid nitrogen only accounts for 38% ~ 50%, and non-essential amino acid such as aspartic acid, content of glutamic acid are more, so protein biological value is lower, but aspartic acid and L-glutamic acid are and are flavor amino acid, so be used for greatly stimulating animal appetite of animal-feed.The main component of molasses is carbohydrate, and it is about 24% ~ 36% that cane molasses contains sucrose, other sugar about 12% ~ 24%; The contained carbohydrate of beet sirup almost is sucrose entirely, about 47% more than.Contain in addition 3% ~ 4% solubility colloid, be mainly xylan, araban and pectin etc.The content of mineral substances of molasses is higher, and is about 8% ~ 10%, but calcium, phosphorus content are not high, and cane molasses is higher than again beet sirup.Potassium, chlorine, sodium, Mg content height in the mineral element, so molasses have laxative qualits.General molasses vitamin contents is low, but pantothenic acid content is higher in the cane molasses, reaches 37mgkg
-1, vitamin H content is also very considerable in addition.With the sugar cane juice of purifying or sugar beet juice inspissation to the massecuite with crystal, with whizzer tell behind the crystallised sugar remaining mother liquor, be " honey ".Also contain volume sucrose in this first molasses, can repeat method and must be second, third molasses etc.Obtain at last a kind of mother liquor, again inspissation crystallization claims waste molasses.What general single title molasses referred to is exactly useless grain honey, can be used as food or feed, also can be used for the raw material of fermentation industry.The present invention utilizes its contained sugar as the carbon source of microorganism growth.
3) monosodium glutamate waste liquid: monosodium glutamate waste liquid is a kind of organic waste water of high density as a kind of waste water of foodstuffs industry, has acid strong, high COD, high BOD, high sulfate radical, high thalline content, does not contain the characteristics of the toxic substances such as heavy metal, belongs to harmful nontoxic waste water.The major cause of its high COD, high BOD is due to L-glutamic acid, residual sugar, SS and the ammonia nitrogen.Total nitrogen is 0.8%～1.0% in the waste liquid, and wherein high-quality nitrogenous source ammonia-state nitrogen accounts for 80%, also contains the nutritive substances such as residual sugar, organic acid, phosphorus, magnesium, potassium.Such as not treated direct discharging, can cause environmental problem, destroy the eubiosis.But during cultivating and produce, the heterotrophism that can be used for microorganism, algae provides the nutritive ingredients such as nitrogenous source.The present invention mainly utilizes its carbon containing nitrogen as the Carbon and nitrogen sources of microorganism growth.
4) sal epsom: sal epsom or magnesium sulfate heptahydrate, molecular formula are MgSO
4(or MgSO
47H
2O), having another name called magnesium sulphate, sal amarum, epsom salt, Epsom salts, is a kind of compound that contains magnesium.Magnesium sulfate heptahydrate easy-weathering in air (drying) is powdery, sloughs gradually crystal water during heating and becomes anhydrous magnesium sulfate.Magnesium has extremely important effect in the metabolism of sugar and protein.Sal epsom is used to a kind of fertilizer in agricultural, because magnesium is one of chlorophyllous main component.Be normally used for the farm crop of potted plant or magnesium deficiency.The present invention mainly utilizes it as the required nutritive element of microorganism growth.
5) potassium primary phosphate: chemical formula is KH
2PO
4, molecular weight is 136.09, is colourless crystallization or white particle sprills.Stable in the air, in the time of 400 ℃, lose water, become metaphosphate.Industrial as buffer reagent, nutrient chemical; Also be used as casate, synthetic seasonings, the raw material of potassium metaphosphate processed, the nutrient chemical of brewer's yeast, reinforcer, raising agent, fermentation assistant.The present invention mainly utilizes it as the buffer reagent in the microorganism culturing, and phosphorus and potassium element are provided.
6) dipotassium hydrogen phosphate: chemical formula is K
2HPO
4, outward appearance is white crystals or amorphous white powder, soluble in water, the aqueous solution is slight alkalinity, is slightly soluble in alcohol, and water absorbability is arranged, self-dissolving when temperature is higher.The intramolecule dehydration was converted into potassium pyrophosphate when relative density was 2.338,204 ℃.The pH value of 1% aqueous solution is 8.9.Be used for medicine, fermentation, microbial culture and produce potassium pyrophosphate etc.Industrial as buffer reagent, nutrient chemical; Also be used as casate, the raw material of potassium metaphosphate processed, the nutrient chemical of brewer's yeast, reinforcer, raising agent, fermentation assistant.The present invention mainly utilizes it as the buffer reagent in the microorganism culturing and nutrient chemical, and phosphorus and potassium element are provided.
(2) application method of Paracoccus denitrificans in cultivation water purifies:
With above-mentioned bacteria concentration through fermentation culture 〉=10
9CFU mL
-1Paracoccus denitrificans bacterium liquid, by 1000 mL(mu Mi Shuishen)
-1Consumption in about at 10 o'clock in day morning August, join the crucian cultivation pond of Yancheng, Jiangsu Province, 20 mu of pond areas, 1.5 meters of the depth of waters are used bacterium liquid 30Kg.Duration of test dissolved oxygen content is at 4.8 ~ 5.2 mgL
-1Between.Test initial ammonia nitrogen, nitrite nitrogen and nitric nitrogen and be respectively 1.55,0.42,0.36 mgL
-110 ~ 12 hours begin to work after using, and nitric nitrogen begins to descend at first, and then nitrite nitrogen and ammonia nitrogen begin to descend.Use rear 20 hours nitrate nitrogen contents to reduce to 0.09 mgL
-1, reduce by 75%; The nitrite nitrogen content is 0.10 mgL after 40 hours
-1, reduce by 76%; Ammonia-nitrogen content was reduced to 0.46 mgL in 60 hours
-1, reduced by 70%.Such water quality situation was kept 7 days always.
Embodiment 2: the cultivation of a kind of Paracoccus denitrificans of the present invention and the application method in cultivation water purifies, adopt following processing step:
(1) cultivation of Paracoccus denitrificans:
Get from the test tube slant ring Paracoccus denitrificans (
Paracoccus denitrificans, ACCC10489, administrative center provides by the preservation of Chinese agriculture microbial strains) lawn, be inoculated in the LB liquid nutrient medium of 100mL sterilization, in 35 ℃, 150 rmin
-1Cultivate 30h on the shaking table.Then with in the LB liquid nutrient medium of its access 50L through sterilizing, in 35 ℃, 150 rmin
-1Cultivate 30h on the shaking table, it is for subsequent use to make the bacterial classification seed liquor.This moment bacterium liquid OD
600Value approximates 1.0, viable count 〉=10
9CFU mL
-1
The liquid fermentation medium that adds cumulative volume 350L in 0.5 ton of stainless steel fermentor tank, cooling is for subsequent use behind 121 ℃ of sterilization 30min.With above-mentioned bacterial classification seed liquor access fermentor tank, be to cultivate 24 hours under the condition of 25% saturated content at 35 ℃, dissolved oxygen content, treat bacterial concentration 〉=10
9CFU mL
-1The time with the phosphoric acid adjust pH be put into after 4.5 through the 25L bucket of sterilization for subsequent use.
Described LB liquid nutrient medium is basic enrichment medium, contains peptone 10 gL
-1, yeast extract paste 5 gL
-1, NaCl 10 gL
-1, regulate pH about 7.0, cooling is for subsequent use behind 121 ℃ of sterilization 20min.
Described liquid fermentation medium, its component ratio is counted by weight: 10 parts in molasses (sugar content 〉=50%), monosodium glutamate waste liquid (COD 〉=80000mgL
-1, TN 〉=16000mgL
-1) 5 parts,
MgSO
47H
20.05 part of O, K
2HPO
41.25 part, KH
2PO
40.6 part, 83.10 parts in water, under the room temperature in fermentor tank dissolving mix, regulate the ratio of molasses and monosodium glutamate waste liquid so that solution C/N mol ratio reaches 11.2, and with NaOH or HCl regulator solution pH value to 7.2,121 ℃ of sterilizations, cooling is rear for subsequent use.
Stainless steel fermentor tank described above is conventional equipment, buys in market, without any particular requirement, adopts common working method.Material of the present invention is convenient sources also, all can buy in market.
(2) application method of Paracoccus denitrificans in cultivation water purifies:
With above-mentioned bacteria concentration through fermentation culture 〉=10
9CFUmL
-1Paracoccus denitrificans bacterium liquid, press 500mL(mu Mi Shuishen)
-1Consumption in about at 10 o'clock in day morning July, join the crucian cultivation pond of Yancheng, Jiangsu Province, 20 mu of pond areas, 1.5 meters of the depth of waters are used bacterium liquid 15Kg.Duration of test dissolved oxygen content is at 6.0 mgL
-1More than.Test initial ammonia nitrogen, nitrite nitrogen and nitric nitrogen and be respectively 1.72,0.38,0.28 mgL
-17 ~ 8 hours begin to work after using, and ammonia nitrogen begins to descend at first, and then nitrite nitrogen and nitric nitrogen begin to descend.Use rear 12 hours ammonia-nitrogen contents to reduce to 0.35mgL
-1, reduce by 80%; Nitrate nitrogen content is reduced to 0.07 mgL after 16 hours
-1, reduce by 75%; 22 hours nitrite nitrogen content to 0.08 mgL
-1, reduced by 79%.Such water quality situation was kept 5 days always.
Embodiment 3: the cultivation of a kind of Paracoccus denitrificans of the present invention and the application method in cultivation water purifies, adopt following processing step:
(1) cultivation of Paracoccus denitrificans:
Get from the test tube slant ring Paracoccus denitrificans (
Paracoccus denitrificans, ACCC10489, administrative center provides by the preservation of Chinese agriculture microbial strains) lawn, be inoculated in the LB liquid nutrient medium of 50mL sterilization, in 30 ℃, 100 rmin
-1Cultivate 24h on the shaking table.Then with in the LB liquid nutrient medium of its access 5L through sterilizing, in 30 ℃, 100 rmin
-1Cultivate 24h on the shaking table, it is for subsequent use to make the bacterial classification seed liquor.This moment bacterium liquid OD
600Value approximates 1.0, viable count 〉=10
9CFU mL
-1
The liquid fermentation medium that adds cumulative volume 700L in 1.0 tons of stainless steel fermentor tanks, cooling is for subsequent use behind 121 ℃ of sterilization 20min.With above-mentioned bacterial classification seed liquor access fermentor tank, be to cultivate 24 hours under the condition of 30% saturated content at 30 ℃, dissolved oxygen content, treat bacterial concentration 〉=10
9CFU mL
-1The time with the phosphoric acid adjust pH be put into after 4.5 through the 25L bucket of sterilization for subsequent use.
Described LB liquid nutrient medium is basic enrichment medium, contains peptone 10 gL
-1, yeast extract paste 5 gL
-1, NaCl 10 gL
-1, regulate pH about 7.0, cooling is for subsequent use behind 121 ℃ of sterilization 20min.
Described liquid fermentation medium, its component ratio is counted by weight: 13 parts in molasses (sugar content 〉=50%), monosodium glutamate waste liquid (COD 〉=80000mgL
-1, TN 〉=16000mgL
-1) 6 parts,
MgSO
47H
20.05 part of O, K
2HPO
41.05 part, KH
2PO
40.3 part, 79.6 parts in water, under the room temperature in fermentor tank dissolving mix, regulate the ratio of molasses and monosodium glutamate waste liquid so that solution C/N mol ratio reaches 11.7, and with NaOH or HCl regulator solution pH value to 7.2,121 ℃ of sterilizations, cooling is rear for subsequent use.
Stainless steel fermentor tank described above is conventional equipment, buys in market, without any particular requirement, adopts common working method.Material of the present invention is convenient sources also, all can buy in market.
(2) application method of Paracoccus denitrificans in cultivation water purifies:
With above-mentioned bacteria concentration through fermentation culture 〉=10
9CFU mL
-1Paracoccus denitrificans bacterium liquid, by 1500 mL(mu Mi Shuishen)
-1Consumption in about at 10 o'clock in day morning June, join the snakeheaded fish cultivating pool in Huzhou, Zhejiang, 15 mu of pond areas, 1.5 meters of the depth of waters are used bacterium liquid 33.75Kg.Dissolved oxygen content is at 2.8 ~ 3.1 mgL during test
-1Between.Test initial ammonia nitrogen, nitrite nitrogen and nitric nitrogen and be respectively 2.08,0.40,0.27 mgL
-1Use to begin to work in rear 24 hours, nitric nitrogen and nitrite nitrogen begin to descend simultaneously, and then ammonia nitrogen falls.Use rear 60 hours nitric nitrogens and nitrite nitrogen content to be 0.08 mgL
-1, reduced by 70% and 80%; Ammonia-nitrogen content is to 0.61mgL after 120 hours
-1, reduced by 71%.Such water quality situation was kept 5 days always.
Claims (6)
1. the cultural method of a Paracoccus denitrificans, it is characterized in that: culturing step is as follows: get Paracoccus denitrificans
Paracoccus denitrificans, ACCC10489 is from Chinese agriculture microbial strains preservation administrative center; Be inoculated in the LB liquid nutrient medium of 20 ~ 100mL sterilization, in 28 ~ 35 ℃, 80 ~ 150 rmin
-1Cultivate 20 ~ 30h on the shaking table; Then with in the LB liquid nutrient medium of its access 3 ~ 50L through sterilizing, in 28 ~ 35 ℃, 80 ~ 150 rmin
-1Cultivate 20 ~ 30h on the shaking table, it is for subsequent use to make the bacterial classification seed liquor;
The liquid fermentation medium that in 0.5 ~ 1.0 ton of stainless steel fermentor tank, adds bacterial classification seed liquor cumulative volume 65% ~ 75%, cooling is for subsequent use behind 115 ~ 121 ℃ of sterilization 15 ~ 30min; With above-mentioned bacterial classification seed liquor access fermentor tank, be to cultivate 24 ~ 30h under the condition of 20% ~ 30% saturated content at 28 ~ 35 ℃, dissolved oxygen content, treat bacterial concentration 〉=10
9CFU mL
-1The time with the phosphoric acid adjust pH be put into after 4 ~ 5 through 1 ~ 25L bucket of sterilization for subsequent use.
2. the cultural method of Paracoccus denitrificans as claimed in claim 1, it is characterized in that: described LB liquid nutrient medium is basic enrichment medium, contains peptone 5 ~ 15 gL
-1, yeast extract paste 3 ~ 8 gL
-1, NaCl 5 ~ 15 gL
-1, regulate pH 6.5-7.5, cooling is for subsequent use behind 115 ~ 121 ℃ of sterilization 15 ~ 30min.
3. the cultural method of Paracoccus denitrificans as claimed in claim 1, it is characterized in that: described liquid fermentation medium, its component ratio is counted by weight: 8 ~ 15 parts in the molasses of sugar content 〉=50%, 4 ~ 10 parts of monosodium glutamate waste liquids, MgSO
47H
20.01 ~ 0.1 part of O, K
2HPO
41.0 ~ 1.5 parts, KH
2PO
40.2 ~ 0.8 part, 78 ~ 88 parts in water;
In dissolving mixing in fermentor tank under the room temperature, the ratio of regulating molasses and monosodium glutamate waste liquid, so that solution C/N mol ratio reaches 10 ~ 15 ︰ 1, and with NaOH or HCl regulator solution pH value to 7.2～7.5,115 ~ 121 ℃ of sterilizations, for subsequent use after the cooling.
4. the cultural method of Paracoccus denitrificans as claimed in claim 3, it is characterized in that: described monosodium glutamate waste liquid COD is 10000 ~ 100000 mgL
-1, TN is 5000 ~ 20000 mgL
-1
5. the cultural method of Paracoccus denitrificans as claimed in claim 1 is characterized in that: the OD of described bacterial classification seed liquor
600Value is 0.8 ~ 1.2, and viable count is 1 * 10
8~ 9 * 10
10CFU mL
-1
6. the Paracoccus denitrificans application in cultivation water purifies is characterized in that: adopt following steps: be 1 * 10 with bacteria concentration
8~ 9 * 10
10CFU mL
-1Paracoccus denitrificans bacterium liquid is by 500 ~ 1500 mL mu Mi Shuishen
-1Consumption o'clock join in the aquaculture water to be evolved in the morning 9 ~ 11; According to practical situation, used once every 5 ~ 10 days.
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