CN105087419B - A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen - Google Patents

A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen Download PDF

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CN105087419B
CN105087419B CN201510090592.5A CN201510090592A CN105087419B CN 105087419 B CN105087419 B CN 105087419B CN 201510090592 A CN201510090592 A CN 201510090592A CN 105087419 B CN105087419 B CN 105087419B
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bacillus subtilis
culture
leather
waste water
strain
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CN105087419A (en
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赵长青
杨秦欢
赵兴秀
张静
邹伟
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四川理工学院
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Abstract

The present invention discloses a kind of bacillus subtilis(Bacillus subtilis)Application in handling leather-making waste water total nitrogen is included the following steps using bacillus subtilis processing leather-making waste water total nitrogen:(l) culture medium is prepared;(2) it ferments:Bacillus subtilis strain is seeded to the shaking flask equipped with culture medium, carries out the zymotic fluid that ferments to obtain;(3) extraction of biological flocculant:Zymotic fluid through centrifugation, extraction, centrifuge again, vacuum freeze drying obtains biological flocculant product;(4) wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, in being stirred on magnetic stirring apparatus.Total nitrogen of the present invention in new leather wastewater treatment by bioflocculant, removal efficiency can reach 81.2%, performance biological flocculant is environmentally protective, does not generate the special performances such as secondary pollution, new method is provided for the high concentration total nitrogen in removal leather-making waste water, it has widened to the application in terms of bacillus subtilis function, there is stronger application value.

Description

A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen

Technical field

The present invention relates to biotechnologies, more particularly to a kind of bacillus subtilis(Bacillus subtilis) Application in handling leather-making waste water total nitrogen.

Background technology

Leather industry is high pollution industry, and with the increase of leather industry yield, the pollutant of leather industry discharge is not yet Disconnected to increase, leather-making waste water environmental pollution problem becomes increasingly conspicuous.

The characteristics of due to leather-making technology, determines that leather-making waste water complicated component, color are deep, and total nitrogen is relatively high.Total nitrogen(TN) Including all nitrogenous compounds in waste water, i.e. organic nitrogen(Protein, polypeptide, amino acid and urea etc.)And inorganic nitrogen(Ammonia nitrogen, Nitrite nitrogen and nitrate nitrogen).It is well known that nitrogen excessive in water body can cause body eutrophication, water quality deterioration is caused, Fish and other biological mortalities;In addition, nitrite can form Carcinogenic Nitrosamines, the life and health of people and animals is endangered;Nitre Hydrochlorate nitrogen can be reduced to nitrite nitrogen;And ammonia nitrogen can pollute air, can also when with waste water irrigation soil containing excessive ammonia nitrogen Crops are caused to be seriously endangered.

Total nitrogen in leather-making waste water is essentially from two aspects:First, to use Inorganic Ammonium in process hides deliming and softening process Salt, at present from the point of view of cost and using effect, there are no can be with the deliming agent of replacing whole inorganic ammonium salt;Another aspect process hides is Using processed collagen fiber --- as the process of primary raw material, a large amount of hide collagen will be hydrolyzed in waste water protein, with useless The ammonification of protein in water, Determination of Total Nitrogen in Waste Water especially ammonia nitrogen concentration increase rapidly, this so that ammonia nitrogen concentration is very high in waste water, reaches To 300-600mg/L, sometimes even waste water occur more handles the higher phenomenon of ammonia nitrogen concentration.National environmental protection portion in What on December 27th, 2013 was formally issued《Process hides and fur manufacturing industrial water pollution object discharge standard》(GB 30486—2013) In, it is clear new to be provided with total nitrogen Con trolling index, it is specified that emission limit to pollutant total nitrogen, to process hides existing enterprise (2014.7.1~2015.12.31), existing enterprise (from 1 day January in 2016), newly-built enterprise and special protection area it is total Nitrogen direct emission limit value is respectively 70,50,50,20mg/L.It can be seen that not allowed to neglect to the improvement of total nitrogen in leather-making waste water Depending on.

For a long time, people do not draw the improvement of total nitrogen in leather-making waste water before compared with the processing paid attention to ammonia nitrogen Play too many concern.At present mainly there is the method for total nitrogen in control leather-making waste water:(1)Cleanly production controls source.In recent years Scholars are dedicated to exploitation and clean deliming and process for tanning, to reduce the content of Determination of Total Nitrogen in Waste Water as far as possible.(2)Biochemical treatment Technology.Researchers explored the different biochemical methods of total nitrogen in removal leather-making waste water, including anaerobic-aerobic in recent years(A/ O)Technique, biofilm reactor(MBR)Method etc..For the discharging standards for enabling the total nitrogen in leather-making waste water to reach new, While using process for cleanly preparing, reducing pollutant total nitrogen from source, need to find effective New Wastewater Treatment Technique.

Invention content

In order to solve the above technical problems, the present invention provides a kind of bacillus subtilis(Bacillus subtilis)'s Using the bacillus subtilis preserving number is CCTCC M 2014512, is preserved in China typical culture collection center, is protected It is on October 26th, 2014 to hide the date, by extracting biological flocculant from Bacillus subtilis strain, the biology is used in combination to wad a quilt with cotton Total nitrogen in solidifying agent processing leather-making waste water, it is environmentally protective, do not generate secondary pollution etc., eliminate the high concentration generated in tanning production Pollution of the total nitrogen waste water to environment, and build total nitrogen in clean leather-making waste water and handle new technology.

Solve a kind of application of bacillus subtilis of the above technical problem, it is characterised in that:Preserving number is CCTCC M 2014512, the bacillus subtilis is applied to reduce total nitrogen in leather-making waste water.

The bacillus subtilis is obtained by cultivating and taming in the activated sludge of tannery.

Steps are as follows for the cultivation and domestication:

(1)The enrichment culture of strain:

The activated sludge of acquisition tannery, which is seeded to, to have sterilized and in the LB culture mediums that cool down, the culture of sludge and culture medium Ratio is volume ratio about 1:8-12 is formulated as follows in 28-32 DEG C of shaking table culture 46-50h, LB culture medium:Peptone 8-12g, ferment Female cream 4-7g, NaCl 8-12g add distilled water to 1000mL, pH6.8-7.2,115-125 DEG C of sterilizing 15-25min;Shaking speed It is 100r/min.

(2)Tamed strain:

5mL enrichment culture bacterium solutions are inoculated in 28-32 DEG C of shaken cultivation in the LB culture mediums that 100mL contains leather-making waste water 45-50h, and the amount for gradually increasing leather-making waste water is tamed step by step to 40 mL, 60 mL, 80 mL;Tame step by step, the purpose is to for The strain of culture is set to can adapt to the high concentration total nitrogen of leather-making waste water.

Pass through step(1)Activated sludge after culture, as enrichment culture bacterium solution.

Whether bacterium colony tames success during domestication, contains leather-making waste water before and after culture when being by detecting the domestication per level-one LB culture mediums nitrogen removal rate whether maintain higher level always depending on, if nitrogen removal rate maintain always it is higher Level can then carry out next stage domestication;It is tamed in aerobic reactor;It is LB culture mediums to tame culture medium all.

3. the separation of strain:

By dilution-plate method, using solid LB media as isolation medium, isolated from the bacterium solution tamed various Bacterium, and each bacterial strain is purified.

Dilution-plate method.I.e.:The activated sludge that 20mL has been tamed is drawn with Sterile pipette, is put into bead Conical flask in, oscillation, the cell of various bacterium is fully dispersed(With micro- sem observation, cell is in unicellular).With sterile suction Pipe therefrom draws 1mL thallus suspension liquids and is added to fill to be mixed well in the Boiling tube of 9mL, then uses aseptic straw from this test tube It draws 1mL to be added in another test tube for filling 9mL sterile waters, be uniformly mixed, and so on be made 10- 1、10- 2、10- 3、10- 4、 10- 5、10- 6、10- 7The thallus suspension liquid of different dilutions.Then the dilution of different dilutions is drawn respectively with Sterile pipette Liquid is in sterile petri dish, then will sterilize and be cooled to 45~50 DEG C or so of LB solid mediums and be poured into each sterile culture In ware, it is about 15mL to be poured into culture medium.Culture dish is gently all around rotated on aseptic operating platform later, makes diluted bacterium Suspension is uniformly mixed with the agar medium of thawing, is stood after mixing.After tablet cooling, tablet is inverted in 37 DEG C of incubators Middle culture 1~2 day.The test tube that the single bacterium colony grown after culture is finally distinguished to picking a little cell inoculation to LB culture mediums is oblique On face.

The step of purifying:The bacterial strain that will be inoculated in each inclined-plane is detached repeatedly by aforementioned dilution-plate method separating step Strain, until separated strain is pure culture, i.e., after lawn is grown, colony characteristics are consistent.

Prioritization scheme also has the screening of bioflocculant-producing bacteria in of the invention, the cultivation and domestication further include biology The screening of bacterium for producing flocculant, such as following steps:

(1)Primary dcreening operation

By the bacillus subtilis strain isolated and purified out be inoculated in flocculation culture medium in cultivate, by culture solution from The heart takes supernatant to carry out the primary dcreening operation of flocculation activity, i.e.,:2 mL culture solutions are taken to be added in 100 mL, 4 g/L Kaolin clay suspensions, It is compareed, is observed with the Kaolin clay suspension for being not added with culture solution simultaneously, whether to occur flocculating to determine whether with flocculation Activity is to carry out primary dcreening operation, if flocculation, preliminary judgement have the ability of generation flocculant, need to further detect in culture solution and wad a quilt with cotton The flocculation activity size of solidifying agent;Flocculation culture medium is formulated as follows:NaNO31-3 g, KC1 0-11 g, K2HPO40.5-1.5 g, MgSO40.3-0.7 g, FeSO40.01 g, sucrose 27-32 g and distilled water 1L remove FeSO4Outside, remaining raw material is in 115- 125 DEG C of sterilizing 15-25 min, after bacterium cooling of having gone out, by FeSO4It is dissolved in the distilled water after sterilizing, filtering, then will filtering FeSO afterwards4Solution is added in the culture medium;

(2)Secondary screening

The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation culture medium and is cultivated, after 45-50h, with wadding The size of solidifying rate produces the size of flocculant ability as bacterial strain is weighed, and therefrom selects the withered grass gemma generated with biological flocculant Bacillus.The bacillus subtilis filtered out can produce flocculant, there is the ability of removal pollutant.

A kind of application of bacillus subtilis in the present invention, it is characterised in that:It is useless using bacillus subtilis processing process hides The method of water total nitrogen includes the following steps:

(l)Culture medium is prepared:NaNO31.5-2.5g KC1 0-11g, K2HPO40.6-1.2 g, MgSO4 0.3-0.7 G, FeSO40.01 g, sucrose 28-32 g and distilled water 1L remove FeSO4Outside, remaining raw material sterilizing 15-25 at 115-125 DEG C Min, after bacterium cooling of having gone out, by FeSO4It is dissolved in the distilled water after sterilizing, with the FeSO of sterilising filter filtering dissolving4, Filtered FeSO is drawn with Sterile pipette4Solution is added in the culture medium;

(2)Fermentation:Bacillus subtilis strain is seeded to the 250mL shaking flasks equipped with 50mL culture mediums, being placed on rotating speed is In the shaking table of 180r/min, the shaken cultivation 45-50h under the conditions of 32-38 DEG C;Strain after culture is inoculated to equipped with 100mL The 250mL conical flasks of culture medium, then 24~68h of shaken cultivation obtains zymotic fluid under the conditions of 32-38 DEG C, strain and culture medium Inoculum concentration ratio is volume ratio 1:8-12;Zymotic fluid refers to step(2)By the culture solution for cultivating 24~68h.

(3)The extraction of biological flocculant:Zymotic fluid is put into the centrifuge that rotating speed is 5000-10000r/min, centrifugation 10min collects supernatant, and the ethyl alcohol of 2-4 times of volume is then added in supernatant, and gained mixture stands 5- at 3-5 DEG C Mixture is put into the centrifuge that rotating speed is 5000~10000r/min again after 7h, centrifuges 8-12min, precipitation is collected, with steaming Distilled water dissolves 3h, lysate is put into the centrifuge that rotating speed is 5000~10000r/min, centrifuges 8-12min, gained precipitation Object with distillation water dissolution 2-3h, is dialysed 1~2 time, until nose can't smell ethanol flavor, to remove small molecule and residual repeatedly again Organic solvent, finally the sediment of centrifugation is placed in vacuum freeze drier and is freeze-dried, can be obtained biological flocculant at Product;

(4)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, it is anti-in being carried out on magnetic stirring apparatus It answers.

The pH 6.7-7.2 of the flocculation culture medium or culture medium.

The step(3)Middle ethyl alcohol is pre-cooled ethanol, 4 DEG C of temperature.

The step(3)The rotating speed of middle centrifuge is 8000r/min.

Bacillus subtilis in the present invention is enriched, domestication, has adapted to the environment containing high concentration total nitrogen waste water, can be A kind of metabolite, as biological flocculant are secreted out of when being cultivated in culture medium;The biological flocculant because mainly contain protein, The macromolecular components such as carbohydrate, glycoprotein, osamine and fat pass through solid suspension the effects that ionic bond, hydrogen bond and in waste water It is combined, while because it has some active groups, overcoming inorganic polymer and Syn-Organic flocculants inherently Defect, can with the pollutant reaction in waste water, achieve the purpose that remove pollutant.Protein, carbohydrate, glycoprotein, osamine and Fat, and these ingredients are all biodegradable, nontoxic, non-secondary pollutions.

The present invention plays biology wadding using the total nitrogen in the leather wastewater treatment by bioflocculant of producing bacillus subtilis life Solidifying agent is environmentally protective, does not generate the special performances such as secondary pollution, has opened the biological flocculant of total nitrogen in removal leather-making waste water New raxa can eliminate pollution of the high concentration total nitrogen waste water generated in tanning production to environment, and it is useless to build clean process hides Total nitrogen handles new technology in water, to provide new method for the high concentration total nitrogen in removal leather-making waste water;In addition, also widening To the application in terms of bacillus subtilis function, stronger application value is made it have.

Specific implementation mode

Below by embodiment, the present invention is described in further detail, but protection scope of the present invention is not only limited in The example.

Embodiment 1

(1)The screening of bacillus subtilis

1. the enrichment culture of strain

The activated sludge that tannery takes is seeded to the LB culture mediums for having sterilized and having cooled down(The culture medium that the present invention mentions is equal By sterilizing and cooling down)In, in 30 DEG C of shaking table culture 48h.LB culture mediums are formulated as follows:Peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, pH 7.0.121 DEG C of sterilizing 20min.

2. tamed strain

5mL enrichment culture bacterium solutions are inoculated in 100mL and contain leather-making waste water(Leather-making waste water 20mL, 2000 degree of total nitrogen(Total nitrogen It is measured using total nitrogen instrument))LB culture mediums in 30 DEG C of shaken cultivation 48h.And the amount of leather-making waste water is gradually increased to 40 ML, 60 mL, 80 mL, are tamed step by step.Its purpose is to so that the strain of culture is can adapt to the high concentration total nitrogen of leather-making waste water.

Whether bacterium colony tames success during domestication, contains leather-making waste water before and after culture when being by detecting the domestication per level-one LB culture mediums nitrogen removal rate whether maintain higher level always depending on, if nitrogen removal rate maintain always it is higher Level can then carry out next stage domestication;It is tamed in aerobic reactor;It is LB culture mediums to tame culture medium all.

3. the separation of strain

By dilution-plate method, using solid LB media as isolation medium, isolated from the bacterium solution tamed various Bacterium, and each bacterial strain is purified.

Dilution-plate method.I.e.:The activated sludge that 20mL has been tamed is drawn with Sterile pipette, is put into bead Conical flask in, oscillation, the cell of various bacterium is fully dispersed(With micro- sem observation, cell is in unicellular).With sterile suction Pipe therefrom draws 1mL thallus suspension liquids and is added to fill to be mixed well in the Boiling tube of 9mL, then uses aseptic straw from this test tube It draws 1mL to be added in another test tube for filling 9mL sterile waters, be uniformly mixed, and so on be made 10- 1、10- 2、10- 3、10- 4、 10- 5、10- 6、10- 7The thallus suspension liquid of different dilutions.Then the dilution of different dilutions is drawn respectively with Sterile pipette Liquid is in sterile petri dish, then will sterilize and be cooled to 45~50 DEG C or so of LB solid mediums and be poured into each sterile culture In ware, it is about 15mL to be poured into culture medium.Culture dish is gently all around rotated on aseptic operating platform later, makes diluted bacterium Suspension is uniformly mixed with the agar medium of thawing, is stood after mixing.After tablet cooling, tablet is inverted in 37 DEG C of incubators Middle culture 1~2 day.The test tube that the single bacterium colony grown after culture is finally distinguished to picking a little cell inoculation to LB culture mediums is oblique On face.

The step of purifying:The bacterial strain that will be inoculated in each inclined-plane is detached repeatedly by aforementioned dilution-plate method separating step Strain, until separated strain is pure culture, i.e., after lawn is grown, colony characteristics are consistent.

4. the screening of bioflocculant-producing bacteria

A. primary dcreening operation

Each bacterial strain isolated and purified out is inoculated in respectively in flocculation culture medium and is cultivated, medium centrifugal takes Clear liquid carries out the primary dcreening operation of flocculation activity, i.e.,:2 mL culture solutions are taken to be added in 100 mL, 4 g/L Kaolin clay suspensions, while with not Add the Kaolin clay suspension of culture solution to be compareed, observe phenomenon, whether to occur flocculating to determine whether having flocculation activity To carry out primary dcreening operation.If flocculation, preliminary judgement have the ability of generation flocculant, need to further detect flocculant in culture solution Flocculation activity size.Flocculation culture medium is formulated as follows:NaNO32 g, KC1 0.5 g, K2HPO41 g, MgSO40.5 g, FeSO40.01 g, 30 g of sucrose and distilled water 1L, pH are naturally, sterilizing.

B. secondary screening

The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation culture medium and is cultivated, after 48h, with flocculating rate Size as the size for weighing bacterial strain and producing flocculant ability, therefrom select the best 1 plant of biological flocculant of flocculating effect and generate Bacterium.

5. the identification of bioflocculant-producing bacteria

Analysis of physio biochemical characteristics is carried out to the higher bioflocculant-producing bacteria of this 1 plant of flocculation activity filtered out, and By Protocols in Molecular Biology, the 16S rRNA gene orders of each bacterial strain are obtained, then are compared by database, its classification letter is obtained Breath determines that its affiliated kind is bacillus subtilis.

(2)Culture medium is prepared:NaNO32 g, KC1 0.5 g, K2HPO41 g, MgSO40.5 g, FeSO40.01 g, Sucrose 30 g and distilled water 1L remove FeSO4Outside, remaining drug 20 min of sterilizing at 121 DEG C will after bacterium cooling of having gone out FeSO4It is dissolved in distilled water after sterilizing.In, with the FeSO of sterilising filter filtering dissolving4, after drawing filtering with Sterile pipette FeSO4Solution is added in the culture medium.

(3)Fermentation

The separated bacillus subtilis screened in this laboratory is seeded to the 250mL equipped with 50mL culture mediums to shake Bottle is placed in the shaking table that rotating speed is 180r/min, under the conditions of 35 DEG C after shaken cultivation 48h, inoculates to equipped with 100mL and cultivate The conical flask of the 250mL of base, then shaken cultivation is for 24 hours under the conditions of 35 DEG C.

(4)The extraction of biological flocculant

Zymotic fluid is put into the centrifuge that rotating speed is 8000r/min, centrifuges 10min, collects supernatant.Then in supernatant The pre-cooled ethanol (4 DEG C) of 3 times of volumes is added in liquid, gained mixture stands 6h at 4 DEG C.Mixture, which is put into rotating speed, is In the centrifuge of 10000r/min, 10min is centrifuged, collects precipitation.With suitable distillation water dissolution 3h, mixture is put into rotating speed In the centrifuge of 8000r/min, to centrifuge 10min, gained sediment with distillation water dissolution 3h, is dialysed 1 time, until nose repeatedly again Until can't smell ethanol flavor, to remove small molecule and remaining organic solvent.The sediment of centrifugation is finally placed in vacuum refrigeration It is freeze-dried in drying machine, biological flocculant finished product can be obtained.

(5)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, it is anti-in being carried out on magnetic stirring apparatus It answers.

0.4~1.0g biological flocculants are added in the beaker equipped with 100mL leather-making waste waters, beaker is then placed in magnetic force It is stirred in blender, 2min is first stirred with the speed of 200r/min, then 10min is stirred with the speed of 50r/min, stand 20min After filter, measure wherein total values of nitrogen might.

When initial total values of nitrogen might is 450mg/L in leather-making waste water, which can remove wherein total nitrogen 365.9mg/ L, removal efficiency reach 81.2%, without secondary pollution.

Embodiment 2

(1)The screening of bacillus subtilis

1. the enrichment culture of strain

The activated sludge that tannery takes is seeded to the LB culture mediums for having sterilized and having cooled down(The culture medium that the present invention mentions is equal By sterilizing and cooling down)In, in 30 DEG C of shaking table culture 48h.LB culture mediums are formulated as follows:Peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL, pH 7.0.121 DEG C of sterilizing 20min.

2. tamed strain

5mL enrichment culture bacterium solutions are inoculated in 100mL and contain leather-making waste water(Leather-making waste water 20mL, 2000 degree of total nitrogen(Total nitrogen It is measured using total nitrogen instrument))LB culture mediums in 30 DEG C of shaken cultivation 48h.And the amount of leather-making waste water is gradually increased to 40 ML, 60 mL, 80 mL, are tamed step by step.Its purpose is to so that the strain of culture is can adapt to the high concentration total nitrogen of leather-making waste water.

3. the separation of strain

By dilution-plate method, using solid LB media as isolation medium, isolated from the bacterium solution tamed various Bacterium, and each bacterial strain is purified.

(2)Culture medium is prepared:NaNO32 g, KC1 0.5 g, K2HPO41 g, MgSO40.5 g, FeSO40.01 g, Sucrose 30 g and distilled water 1L remove FeSO4Outside, remaining drug 20 min of sterilizing at 121 DEG C will after bacterium cooling of having gone out FeSO4It is dissolved in distilled water after sterilizing.In, with the FeSO of sterilising filter filtering dissolving4, after drawing filtering with Sterile pipette FeSO4Solution is added in the culture medium;

(3)Fermentation

The separated bacillus subtilis screened in this laboratory is seeded to the 250mL equipped with 50mL culture mediums to shake Bottle is placed in the shaking table that rotating speed is 180r/min, under the conditions of 35 DEG C after shaken cultivation 48h, inoculates to equipped with 100mL and cultivate The conical flask of the 250mL of base, then shaken cultivation is for 24 hours under the conditions of 35 DEG C.

(4)The extraction of biological flocculant

Zymotic fluid is put into the centrifuge that rotating speed is 8000r/min, centrifuges 10min, collects supernatant.Then in supernatant The pre-cooled ethanol (4 DEG C) of 3 times of volumes is added in liquid, gained mixture stands 6h at 4 DEG C.Mixture, which is put into rotating speed, is In the centrifuge of 10000r/min, 10min is centrifuged, collects precipitation.With suitable distillation water dissolution 3h, mixture is put into rotating speed In the centrifuge of 8000r/min, to centrifuge 10min, gained sediment with distillation water dissolution 3h, is dialysed 1 time, until nose repeatedly again Until can't smell ethanol flavor, to remove small molecule and remaining organic solvent.The sediment of centrifugation is finally placed in vacuum refrigeration It is freeze-dried in drying machine, biological flocculant finished product can be obtained.

(5)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, it is anti-in being carried out on magnetic stirring apparatus It answers.

0.4~1.0g biological flocculants are added in the beaker equipped with 100mL leather-making waste waters, beaker is then placed in magnetic force It is stirred in blender, 2min is first stirred with the speed of 200r/min, then 10min is stirred with the speed of 50r/min, stand 20min After filter, measure wherein total values of nitrogen might.

When initial total values of nitrogen might is 450mg/L in leather-making waste water, which can remove wherein total nitrogen 348.75mg/ L, removal efficiency reach 77.5%, and degradation speed is fast, without secondary pollution.

Embodiment 3

The screening of bacillus subtilis

(1)The enrichment culture of strain:

The activated sludge of acquisition tannery, which is seeded to, to have sterilized and in the LB culture mediums that cool down, the culture of sludge and culture medium Ratio is volume ratio about 1:8, it is formulated as follows in 28 DEG C of shaking table culture 46h, LB culture mediums:Peptone 8g, yeast extract 4g, NaCl 8g adds distilled water to 1000mL, pH6.8,115 DEG C of sterilizing 25min;Shaking speed is 100r/min.

(2)Tamed strain:

5mL enrichment culture bacterium solutions are inoculated in 28 DEG C of shaken cultivation 50h in the LB culture mediums that 100mL contains leather-making waste water, And the amount for gradually increasing leather-making waste water is tamed step by step to 40 mL, 60 mL, 80 mL;It tames step by step, its purpose is to make training Foster strain can adapt to the high concentration total nitrogen of leather-making waste water.

Pass through step(1)Activated sludge after culture, as enrichment culture bacterium solution.

3. the separation of strain:

By dilution-plate method, using solid LB media as isolation medium, isolated from the bacterium solution tamed various Bacterium, and each bacterial strain is purified.

The screening of bioflocculant-producing bacteria

(1)Primary dcreening operation

By the bacillus subtilis strain isolated and purified out be inoculated in flocculation culture medium in cultivate, by culture solution from The heart takes supernatant to carry out the primary dcreening operation of flocculation activity, i.e.,:2 mL culture solutions are taken to be added in 100 mL, 4 g/L Kaolin clay suspensions, It is compareed, is observed with the Kaolin clay suspension for being not added with culture solution simultaneously, whether to occur flocculating to determine whether with flocculation Activity is to carry out primary dcreening operation, if flocculation, preliminary judgement have the ability of generation flocculant, need to further detect in culture solution and wad a quilt with cotton The flocculation activity size of solidifying agent;Flocculation culture medium is formulated as follows:NaNO31 g, KC1 0 g, K2HPO40.5 g, MgSO4 0.3g, FeSO40.01 g, sucrose 27 g and distilled water 1L remove FeSO4Outside, remaining raw material 25 min of sterilizing at 115 DEG C, are waited for After bacterium of having gone out cooling, by FeSO4It is dissolved in the distilled water after sterilizing, filtering, then by filtered FeSO4The training is added in solution It supports in base, flocculate medium pH 6.7;

(2)Secondary screening

The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation culture medium and is cultivated, after 45-50h, with wadding The size of solidifying rate produces the size of flocculant ability as bacterial strain is weighed, and therefrom selects the withered grass gemma generated with biological flocculant Bacillus.The bacillus subtilis filtered out can produce flocculant, there is the ability of removal pollutant.

Included the following steps using the method that bacillus subtilis handles leather-making waste water total nitrogen:

(l)Culture medium is prepared:NaNO31.5g, KC1 0g, K2HPO40.6 g, MgSO40.3 g, FeSO40.01 g, Sucrose 28 g and distilled water 1L remove FeSO4Outside, remaining raw material 25 min of sterilizing at 115 DEG C will after bacterium cooling of having gone out FeSO4It is dissolved in the distilled water after sterilizing, with the FeSO of sterilising filter filtering dissolving4, after drawing filtering with Sterile pipette FeSO4Solution is added in the culture medium, medium pH 6.7;

(2)Fermentation:Bacillus subtilis strain is seeded to the 250mL shaking flasks equipped with 50mL culture mediums, being placed on rotating speed is In the shaking table of 180r/min, the shaken cultivation 45h under the conditions of 32-38 DEG C;Strain after culture, which is inoculated to equipped with 100mL, to be cultivated The 250mL conical flasks of base, then shaken cultivation obtains zymotic fluid, the inoculum concentration ratio of strain and culture medium for 24 hours under the conditions of 32 DEG C It is volume ratio 1:8;Zymotic fluid refers to step(2)By cultivating culture solution for 24 hours.

(3)The extraction of biological flocculant:Zymotic fluid is put into the centrifuge that rotating speed is 10000r/min, centrifuges 10min, Supernatant is collected, the pre-cooled ethanol of 4 times of volumes is then added in supernatant, 4 DEG C of temperature, gained mixture is stood at 5 DEG C Mixture is put into the centrifuge that rotating speed is 10000r/min again after 5h, centrifuges 12min, collects precipitation, with distillation water dissolution Lysate is put into the centrifuge that rotating speed is 10000r/min by 3h, centrifuges 12min, gained sediment is again with distillation water dissolution 3h dialyses 2 times repeatedly,, finally will centrifugation to remove small molecule and remaining organic solvent until nose can't smell ethanol flavor Sediment be placed in vacuum freeze drier and be freeze-dried, biological flocculant finished product can be obtained;

(4)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, it is anti-in being carried out on magnetic stirring apparatus It answers.

0.4~1.0g biological flocculants are added in the beaker equipped with 100mL leather-making waste waters, beaker is then placed in magnetic force It is stirred in blender, 2min is first stirred with the speed of 200r/min, then 10min is stirred with the speed of 50r/min, stand 20min After filter, measure wherein total values of nitrogen might.

When initial total values of nitrogen might is 580mg/L in leather-making waste water, which can remove wherein total nitrogen 436.74mg/ L, removal efficiency reach 75.3%, and degradation speed is fast, without secondary pollution.

Embodiment 4

The screening of bacillus subtilis

(1)The enrichment culture of strain:

The activated sludge of acquisition tannery, which is seeded to, to have sterilized and in the LB culture mediums that cool down, the culture of sludge and culture medium Ratio is volume ratio about 1:12, it is formulated as follows in 32 DEG C of shaking table culture 46h, LB culture mediums:Peptone 12g, yeast extract 7g, NaC12g adds distilled water to 1000mL, pH7.2,125 DEG C of sterilizing 25min;Shaking speed is 100r/min.

(2)Tamed strain:

5mL enrichment culture bacterium solutions are inoculated in 28-32 DEG C of shaken cultivation in the LB culture mediums that 100mL contains leather-making waste water 45-50h, and the amount for gradually increasing leather-making waste water is tamed step by step to 40 mL, 60 mL, 80 mL;Tame step by step, the purpose is to for The strain of culture is set to can adapt to the high concentration total nitrogen of leather-making waste water.

Pass through step(1)Activated sludge after culture, as enrichment culture bacterium solution.

3. the separation of strain:

By dilution-plate method, using solid LB media as isolation medium, isolated from the bacterium solution tamed various Bacterium, and each bacterial strain is purified.

The screening of bioflocculant-producing bacteria

(1)Primary dcreening operation

By the bacillus subtilis strain isolated and purified out be inoculated in flocculation culture medium in cultivate, by culture solution from The heart takes supernatant to carry out the primary dcreening operation of flocculation activity, i.e.,:2 mL culture solutions are taken to be added in 100 mL, 4 g/L Kaolin clay suspensions, It is compareed, is observed with the Kaolin clay suspension for being not added with culture solution simultaneously, whether to occur flocculating to determine whether with flocculation Activity is to carry out primary dcreening operation, if flocculation, preliminary judgement have the ability of generation flocculant, need to further detect in culture solution and wad a quilt with cotton The flocculation activity size of solidifying agent;Flocculation culture medium is formulated as follows:NaNO33 g, KC1 1 g, K2HPO41.5 g, MgSO4 0.7 G, FeSO40.01 g, sucrose 32 g and distilled water 1L remove FeSO4Outside, remaining raw material 25 min of sterilizing at 125 DEG C, wait going out After complete bacterium cooling, by FeSO4It is dissolved in the distilled water after sterilizing, filtering, then by filtered FeSO4The culture is added in solution In base, flocculate medium pH 7.2;

(2)Secondary screening

The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation culture medium and is cultivated, after 50h, with flocculation The size of rate produces the size of flocculant ability as bacterial strain is weighed, and therefrom selects the bacillus subtilis generated with biological flocculant Bacterium.The bacillus subtilis filtered out can produce flocculant, there is the ability of removal pollutant.

Included the following steps using the method that bacillus subtilis handles leather-making waste water total nitrogen:

(l)Culture medium is prepared:NaNO32.5g, KC1 1g, K2HPO41.2 g, MgSO40.7 g, FeSO40.01 g, Sucrose 32 g and distilled water 1L remove FeSO4Outside, remaining raw material 25 min of sterilizing at 125 DEG C will after bacterium cooling of having gone out FeSO4It is dissolved in the distilled water after sterilizing, with the FeSO of sterilising filter filtering dissolving4, after drawing filtering with Sterile pipette FeSO4Solution is added in the culture medium, medium pH 7.2;

(2)Fermentation:Bacillus subtilis strain is seeded to the 250mL shaking flasks equipped with 50mL culture mediums, being placed on rotating speed is In the shaking table of 180r/min, the shaken cultivation 50h under the conditions of 38 DEG C;Strain after culture is inoculated to equipped with 100mL culture mediums 250mL conical flasks, then shaken cultivation 68h obtains zymotic fluid under the conditions of 38 DEG C, and the inoculum concentration ratio of strain and culture medium is Volume ratio 1: 12;Zymotic fluid refers to step(2)By the culture solution for cultivating 68h.

(3)The extraction of biological flocculant:Zymotic fluid is put into the centrifuge that rotating speed is 5000r/min, centrifuges 10min, Supernatant is collected, the pre-cooled ethanol of 2 times of volumes is then added in supernatant, 4 DEG C of temperature, gained mixture is stood at 3 DEG C Mixture is put into the centrifuge that rotating speed is 5000r/min again after 5-7h, centrifuges 8min, collects precipitation, with distillation water dissolution Lysate is put into the centrifuge that rotating speed is 5000r/min by 3h, centrifuges 8min, and gained sediment uses distillation water dissolution 2h again, It dialyses 1 time repeatedly, until nose can't smell ethanol flavor, to remove small molecule and remaining organic solvent, finally by centrifugation Sediment is placed in vacuum freeze drier and is freeze-dried, and biological flocculant finished product can be obtained;

(4)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, it is anti-in being carried out on magnetic stirring apparatus It answers.

0.4~1.0g biological flocculants are added in the beaker equipped with 100mL leather-making waste waters, beaker is then placed in magnetic force It is stirred in blender, 2min is first stirred with the speed of 200r/min, then 10min is stirred with the speed of 50r/min, stand 20min After filter, measure wherein total values of nitrogen might.

When initial total values of nitrogen might is 400mg/L in leather-making waste water, which can remove wherein total nitrogen 330.4mg/ L, removal efficiency reach 82.6%, and degradation speed is fast, without secondary pollution.

The invention is not limited in specific implementation modes above-mentioned.The present invention, which expands to, any in the present specification to be disclosed New feature or any new combination, and disclose any new method or process the step of or any new combination.

Claims (6)

1. a kind of bacillus subtilis(Bacillus subtilis)Application, it is characterised in that:The bacillus subtilis Its preserving number is CCTCC M 2014512, is applied to reduce total nitrogen in leather-making waste water;The bacillus subtilis is by tannery It is cultivated in activated sludge and tames and obtain.
2. a kind of bacillus subtilis according to claim 1(Bacillus subtilis)Application, feature exists In:Steps are as follows for the cultivation and domestication:(1)The enrichment culture of strain:
The activated sludge of acquisition tannery, which is seeded to, to have sterilized and in the LB culture mediums that cool down, the culture ratio of sludge and culture medium It is volume ratio about 1:8-12 is formulated as follows in 28-32 DEG C of shaking table culture 46-50h, LB culture medium:Peptone 8-12g, yeast extract 4-7g, NaCl 8-12g add distilled water to 1000mL, pH6.8-7.2,115-125 DEG C of sterilizing 15-25min;
(2)Tamed strain:
5mL enrichment culture bacterium solutions are inoculated in 28-32 DEG C of shaken cultivation 45- in the LB culture mediums that 100mL contains leather-making waste water 50h, and the amount for gradually increasing leather-making waste water is tamed step by step to 40 mL, 60 mL, 80 mL;
(3)The separation of strain:
By dilution-plate method, using solid LB media as isolation medium, various bacteriums are isolated from the bacterium solution tamed, And each bacterial strain is purified, obtain Bacillus subtilis strain.
3. according to a kind of bacillus subtilis described in claim 2(Bacillus subtilis)Application, feature exists In:The cultivation and domestication further include the screening of bioflocculant-producing bacteria, such as following steps:
(1)Primary dcreening operation
The bacillus subtilis strain isolated and purified out is inoculated in flocculation culture medium and is cultivated, medium centrifugal takes Supernatant carries out the primary dcreening operation of flocculation activity, i.e.,:2 mL culture solutions are taken to be added in 100 mL, 4 g/L Kaolin clay suspensions, while with The Kaolin clay suspension for being not added with culture solution is compareed, observation, with whether occur flocculating to determine whether with flocculation activity from And primary dcreening operation is carried out, if flocculation, preliminary judgement has the ability of generation flocculant;Flocculation culture medium is formulated as follows:NaNO3 1-3 G, KC1 0-11 g, K2HPO40.5-1.5 g, MgSO40.3-0.7 g, FeSO40.01 g, sucrose 27-32 g and distilled water 1L removes FeSO4Outside, remaining raw material sterilizing 15-25 min at 115-125 DEG C, after bacterium cooling of having gone out, by FeSO4It is dissolved in In distilled water after sterilizing, filtering, then by filtered FeSO4Solution is added in the culture medium;The flocculation medium pH 6.7-7.2;
(2)Secondary screening
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation culture medium and is cultivated, after 45-50h, with flocculating rate Size as weigh bacterial strain produce flocculant ability size, therefrom select the bacillus subtilis generated with biological flocculant Bacterium.
4. a kind of bacillus subtilis according to any one of claim 1-3(Bacillus subtilis)Application, It is characterized in that:Included the following steps using the method that bacillus subtilis handles leather-making waste water total nitrogen:
(l)Culture medium is prepared:NaNO31.5-2.5g KC1 0-11g, K2HPO40.6-1.2 g, MgSO40.3-0.7 g, FeSO40.01 g, sucrose 28-32 g and distilled water 1L remove FeSO4Outside, remaining raw material sterilizing 15-25 at 115-125 DEG C Min, after bacterium cooling of having gone out, by FeSO4It is dissolved in the distilled water after sterilizing, filtering, then by filtered FeSO4Solution adds Enter in the culture medium;
(2)Fermentation:Bacillus subtilis strain is seeded to the 250mL shaking flasks equipped with 50mL culture mediums, it is 180r/ to be placed on rotating speed In the shaking table of min, the shaken cultivation 45-50h under the conditions of 32-38 DEG C;Strain after culture is inoculated to equipped with 100mL culture mediums 250mL conical flasks, then 24~68h of shaken cultivation obtains zymotic fluid, the inoculum concentration of strain and culture medium under the conditions of 32-38 DEG C Ratio is volume ratio 1:8-12;The pH 6.7-7.2 of the culture medium;
(3)The extraction of biological flocculant:Zymotic fluid is put into the centrifuge that rotating speed is 5000-10000r/min, centrifugation 10min collects supernatant, and the ethyl alcohol of 2-4 times of volume is then added in supernatant, and gained mixture stands 5- at 3-5 DEG C Mixture is put into the centrifuge that rotating speed is 5000~10000r/min again after 7h, centrifuges 8-12min, precipitation is collected, with steaming Distilled water dissolves 2.5-3.5h, lysate is put into the centrifuge that rotating speed is 5000~10000r/min, centrifuges 8-12min, institute Sediment is obtained again with distillation water dissolution 2-3h, is dialysed 1~2 time repeatedly, then the sediment of centrifugation is placed in vacuum freeze drier Middle freeze-drying obtains biological flocculant finished product;
(4)Wastewater treatment:Biological flocculant is added in the beaker equipped with leather-making waste water, in being reacted on magnetic stirring apparatus.
5. according to a kind of bacillus subtilis described in claim 4(Bacillus subtilis)Application, feature exists In:The step(3)Middle ethyl alcohol is pre-cooled ethanol, 3-5 DEG C of temperature.
6. according to a kind of bacillus subtilis described in claim 4(Bacillus subtilis)Application, feature exists In:The step(3)The rotating speed of middle centrifuge is 8000r/min.
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