CN105087419A - Application of bacillus subtilis in treating total nitrogen in tannery wastewater - Google Patents
Application of bacillus subtilis in treating total nitrogen in tannery wastewater Download PDFInfo
- Publication number
- CN105087419A CN105087419A CN201510090592.5A CN201510090592A CN105087419A CN 105087419 A CN105087419 A CN 105087419A CN 201510090592 A CN201510090592 A CN 201510090592A CN 105087419 A CN105087419 A CN 105087419A
- Authority
- CN
- China
- Prior art keywords
- substratum
- subtilis
- flocculation
- feso
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Separation Of Suspended Particles By Flocculating Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of bacillus subtilis in treating total nitrogen in tannery wastewater. The application comprises the following steps: (1) preparing a culture medium; (2) fermenting, namely, inoculating bacillus subtilis strain into a shake flask filled with the culture medium, and fermenting to obtain a fermentation broth; (3) extracting a biological flocculant, namely, carrying out centrifugation, extraction, second-time centrifugation and vacuum freeze drying to obtain the biological flocculant; and (4) carrying out wastewater treatment, namely, placing the biological flocculant into a beaker filled with tannery wastewater, placing the beaker on a magnetic stirrer, and carrying stirring treatment. According to the application, the novel method of treating the total nitrogen in tannery wastewater by adopting the biological flocculant is adopted, and the removal efficiency can achieve 81.2, so that the unique properties of no pollution, no generation of secondary pollution and the like of the biological flocculant are fully exerted; a novel method is provided for removing the high-concentration total nitrogen in tannery wastewater; the application of bacillus subtilis in the aspect of function is expanded; and therefore, the application value is high.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of subtilis (
bacillussubtilis) processing the application in leather-making waste water total nitrogen.
Background technology
Leather industry is high pollution industry, and along with the increase of leather industry output, the pollutent of tanning industry discharge is also in continuous increase, and leather-making waste water environmental pollution problem becomes increasingly conspicuous.
Due to leather-making technology, determine leather-making waste water complicated component, color is dark, total nitrogen is higher.Total nitrogen (TN) comprises all nitrogenous compounds in waste water, i.e. organonitrogen (protein, polypeptide, amino acid and urea etc.) and inorganic nitrogen (ammonia nitrogen, nitrite nitrogen and nitrate nitrogen).As everyone knows, nitrogen too much in water body can cause body eutrophication, causes water quality deterioration, fish and other biological mortality; In addition, nitrite can form Carcinogenic Nitrosamines, the life and health of harm people and animals; Nitrate nitrogen can be reduced to nitrite nitrogen; And ammonia nitrogen meeting atmosphere pollution, use during the waste water irrigation soil containing excessive ammonia nitrogen and farm crop also can be caused to be subject to serious harm.
Total nitrogen in leather-making waste water is mainly from two aspects: one is will use inorganic ammonium salt in process hides deliming and softening process, and at present from cost and result of use, also not having can the deliming agent of replacing whole inorganic ammonium salt; Process hides is with processed collagen fiber on the other hand---protein is the process of main raw material, a large amount of hide collagens will be hydrolyzed in waste water, along with the ammonification of protein in waste water, Determination of Total Nitrogen in Waste Water particularly ammonia nitrogen concentration raises rapidly, this makes ammonia nitrogen concentration in waste water very high, reach 300-600mg/L, sometimes even occur that waste water more processes the higher phenomenon of ammonia nitrogen concentration.In national environmental protection portion in " process hides and fur manufacturing industrial water pollution thing emission standard " (GB30486-2013) of formally issuing on December 27th, 2013; clearly newly be provided with total nitrogen Con trolling index; define the emission limit to pollutent total nitrogen, 70 be respectively to the direct emission limit of total nitrogen in process hides existing enterprise (2014.7.1 ~ 2015.12.31), existing enterprise's (from 1 day January in 2016), newly-built enterprise and special protection area, 50,50,20mg/L.As can be seen here, the improvement of total nitrogen in leather-making waste water be can not be ignored.
For a long time, people comparatively pay attention to the process to ammonia nitrogen, are not causing too many concern before to the improvement of total nitrogen in leather-making waste water.In current control leather-making waste water, the method for total nitrogen mainly contains: (1) cleanly production, namely controls source.Scholars were devoted to exploitation and cleaned deliming and process for tanning, to reduce the content of Determination of Total Nitrogen in Waste Water as far as possible in recent years.(2) biochemical processing.Investigators explored the different biochemical methods removing total nitrogen in leather-making waste water in recent years, comprised anaerobic-aerobic (A/O) technique, biofilm reactor (MBR) method etc.In order to the discharging standards making the total nitrogen in leather-making waste water can reach new, while adopting process for cleanly preparing, reducing pollutent total nitrogen from source, need to find effective New Wastewater Treatment Technique.
Summary of the invention
In order to solve above technical problem, the invention provides a kind of subtilis (
bacillussubtilis) application, described subtilis preserving number is CCTCCM2014512, be preserved in China typical culture collection center, preservation date is on October 26th, 2014, by extracting biological flocculant from Bacillus subtilis strain, and with the total nitrogen in this leather wastewater treatment by bioflocculant, environmental protection, do not produce secondary pollution etc., the high density total nitrogen waste water produced in elimination tanning production to the pollution of environment, and builds total nitrogen process new technology in clean leather-making waste water.
Solve the application of a kind of subtilis of above technical problem, it is characterized in that: preserving number is CCTCCM2014512, described subtilis is applied to and reduces total nitrogen in leather-making waste water.
Described subtilis is by cultivating in the active sludge of tannery and taming and obtain.
Described cultivation and domestication step as follows:
(1) enrichment culture of bacterial classification:
Gather the active sludge of tannery and be seeded to sterilizing and in the LB substratum of cooling, the cultivation ratio of mud and substratum is that volume ratio is about 1:8-12,46-50h is cultivated in 28-32 DEG C of shaking table, LB substratum is formulated as follows: peptone 8-12g, yeast extract paste 4-7g, NaCl8-12g, adding distil water is to 1000mL, pH6.8-7.2,115-125 DEG C of sterilizing 15-25min; Shaking speed is 100r/min.
(2) tamed strain:
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 28-32 DEG C of shaking culture 45-50h in the LB substratum of leather-making waste water, and the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step; Taming step by step, its objective is the high density total nitrogen in order to enable the bacterial classification of cultivation adapt to leather-making waste water.
Active sludge after being cultivated by step (1), is enrichment culture bacterium liquid.
In domestication process, whether bacterium colony tames successfully, whether the nitrogen removal rate of LB substratum containing leather-making waste water before and after cultivating when being by detecting the domestication of every one-level maintains higher level and fixed all the time, if nitrogen removal rate maintains higher level all the time, then next stage domestication can be carried out; Tame in aerobic reactor; Domestication substratum is all LB substratum.
3. the separation of bacterial classification:
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain.
Dilution-plate method.That is: with the active sludge that Sterile pipette absorption 20mL has tamed, put into the Erlenmeyer flask with granulated glass sphere, vibration, the cell of various bacterium is fully disperseed (with microscopic examination, cell is unicellular).Therefrom draw 1mL thallus suspension liquid with aseptic straw to add in the Boiling tube filling 9mL and fully mix, then add another fill in the test tube of 9mL sterilized water with drawing 1mL in aseptic straw from then on test tube, mix, the rest may be inferred makes 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7different dilution thallus suspension liquid.Then draw different dilution diluent respectively in sterile petri dish with Sterile pipette, then by sterilizing and the LB solid medium being cooled to about 45 ~ 50 DEG C is poured in each sterile petri dish, impouring substratum is about 15mL.Afterwards culture dish is all around rotated gently on aseptic operating platform, the bacteria suspension of dilution is mixed with the nutrient agar of thawing, leave standstill after mixing.After dull and stereotyped cooling, flat-plate inverted is placed in 37 DEG C of incubators and cultivates 1 ~ 2 day.Finally by the single bacterium colony that grows after cultivating respectively a little cell of picking be inoculated on the test tube slant of LB substratum.
The step of purifying: by the bacterial strain inoculated in each inclined-plane, by aforementioned dilution-plate method separating step, is separated bacterial classification repeatedly, until the bacterial classification be separated is pure culture, after namely lawn grows, its colony characteristics unanimously.
Prioritization scheme, also has the screening of bioflocculant-producing bacteria in the present invention, described cultivation and domestication also comprise the screening of bioflocculant-producing bacteria, as following steps:
(1) primary dcreening operation
Bacillus subtilis strain separation and purification gone out is inoculated in flocculation substratum and cultivates, by medium centrifugal, get the primary dcreening operation that supernatant liquor carries out flocculation activity, that is: getting 2mL nutrient solution adds in 100mL4g/L Kaolin clay suspension, contrast with the Kaolin clay suspension not adding nutrient solution simultaneously, observe, whether there is flocculation to judge whether there is flocculation activity thus to carry out primary dcreening operation, if flocculation, then preliminary judgement has the ability of producing biological flocculant, needs the flocculation activity size detecting flocculation agent in nutrient solution further; Flocculation substratum is formulated as follows: NaNO
31-3g, KC10-11g, K
2hPO
40.5-1.5g, MgSO
40.3-0.7g, FeSO
40.01g, sucrose 27-32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115-125 DEG C of sterilizing 15-25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter, then by the FeSO after filtration
4solution adds in this substratum;
(2) multiple sieve
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation substratum and cultivates, after 45-50h, using the size of flocculating rate as the size weighing bacterial strain produce flocculant ability, therefrom select the subtilis that there is biological flocculant and produce.The subtilis filtered out can produce flocculant, has the ability removing pollutent.
The application of a kind of subtilis in the present invention, is characterized in that: utilize the method for subtilis process leather-making waste water total nitrogen to comprise the following steps:
L () substratum is prepared: NaNO
31.5-2.5g, KC10-11g, K
2hPO
40.6-1.2g, MgSO
40.3-0.7g, FeSO
40.01g, sucrose 28-32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115-125 DEG C of sterilizing 15-25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter the FeSO dissolved with sterilising filter
4, draw the FeSO after filtering with Sterile pipette
4solution adds in this substratum;
(2) ferment: Bacillus subtilis strain is seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, shaking culture 45-50h under 32-38 DEG C of condition; Bacterial classification after cultivation inoculates the 250mL Erlenmeyer flask to being equipped with 100mL substratum, and then under 32-38 DEG C of condition, shaking culture 24 ~ 68h obtains fermented liquid, and the inoculum size ratio of bacterial classification and substratum is volume ratio 1:8-12; Fermented liquid refers to the nutrient solution of step (2) through cultivation 24 ~ 68h.
(3) extraction of biological flocculant: fermented liquid is put into the whizzer that rotating speed is 5000-10000r/min, centrifugal 10min, collect supernatant liquor, then in supernatant liquor, add the ethanol of 2-4 times of volume, mixture is put into the whizzer that rotating speed is 5000 ~ 10000r/min leave standstill 5-7h at 3-5 DEG C after by gained mixture again, centrifugal 8-12min, collecting precipitation, 3h is dissolved with distilled water, lysate is put into the whizzer that rotating speed is 5000 ~ 10000r/min, centrifugal 8-12min, gained throw out dissolves 2-3h with distilled water again, repeatedly dialyse 1 ~ 2 time, to nose can't smell ethanol taste, to remove small molecules and residual organic solvent, finally centrifugal throw out is placed in vacuum freeze drier lyophilize, biological flocculant finished product can be obtained,
(4) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
The pH6.7-7.2 of described flocculation substratum or substratum.
In described step (3), ethanol is pre-cooled ethanol, temperature 4 DEG C.
In described step (3), the rotating speed of whizzer is 8000r/min.
Subtilis in the present invention, through enrichment, domestication, has adapted to the environment containing high density total nitrogen waste water, has secreted a kind of meta-bolites, be biological flocculant when can cultivate in the medium; This biological flocculant contains the macromolecular components such as protein, carbohydrate, glycoprotein, osamine and fat because of main, combined with the suspended solid in waste water by the effect such as ionic linkage, hydrogen bond, because of it, there are some active groups simultaneously, overcome inorganic polymer and Syn-Organic flocculants defect inherently, with the pollutant reaction in waste water, the object removing pollutent can be reached.Protein, carbohydrate, glycoprotein, osamine and fat, and these compositions are all biodegradable, nontoxic, non-secondary pollution.
Total nitrogen in the leather wastewater treatment by bioflocculant that the present invention utilizes producing bacillus subtilis raw, play biological flocculant environmental protection, do not produce the special performances such as secondary pollution, open the biological flocculant New raxa removing total nitrogen in leather-making waste water, the high density total nitrogen waste water that produces in tanning production can be eliminated to the pollution of environment, and build total nitrogen process new technology in clean leather-making waste water, thus be that the high density total nitrogen removed in leather-making waste water provides novel method; In addition, also widen the application to subtilis function aspects, make it have stronger using value.
Embodiment
Below by embodiment, the present invention is described in further detail, but protection scope of the present invention has more than and is limited to this example.
Embodiment 1
(1) screening of subtilis
1. the enrichment culture of bacterial classification
Active sludge tannery got is seeded to sterilizing and in the LB substratum of cooling (substratum that the present invention mentions all be through sterilizing and cooling), cultivate 48h in 30 DEG C of shaking tables.LB substratum is formulated as follows: peptone 10g, yeast extract paste 5g, NaCl10g, distilled water 1000mL, pH7.0.121 DEG C of sterilizing 20min.
2. tamed strain
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 30 DEG C of shaking culture 48h in the LB substratum of leather-making waste water (leather-making waste water 20mL, total nitrogen 2000 degree (total nitrogen adopts total nitrogen instrument to measure)).And the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step.Its objective is the high density total nitrogen in order to enable the bacterial classification of cultivation adapt to leather-making waste water.
In domestication process, whether bacterium colony tames successfully, whether the nitrogen removal rate of LB substratum containing leather-making waste water before and after cultivating when being by detecting the domestication of every one-level maintains higher level and fixed all the time, if nitrogen removal rate maintains higher level all the time, then next stage domestication can be carried out; Tame in aerobic reactor; Domestication substratum is all LB substratum.
3. the separation of bacterial classification
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain.
Dilution-plate method.That is: with the active sludge that Sterile pipette absorption 20mL has tamed, put into the Erlenmeyer flask with granulated glass sphere, vibration, the cell of various bacterium is fully disperseed (with microscopic examination, cell is unicellular).Therefrom draw 1mL thallus suspension liquid with aseptic straw to add in the Boiling tube filling 9mL and fully mix, then add another fill in the test tube of 9mL sterilized water with drawing 1mL in aseptic straw from then on test tube, mix, the rest may be inferred makes 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7different dilution thallus suspension liquid.Then draw different dilution diluent respectively in sterile petri dish with Sterile pipette, then by sterilizing and the LB solid medium being cooled to about 45 ~ 50 DEG C is poured in each sterile petri dish, impouring substratum is about 15mL.Afterwards culture dish is all around rotated gently on aseptic operating platform, the bacteria suspension of dilution is mixed with the nutrient agar of thawing, leave standstill after mixing.After dull and stereotyped cooling, flat-plate inverted is placed in 37 DEG C of incubators and cultivates 1 ~ 2 day.Finally by the single bacterium colony that grows after cultivating respectively a little cell of picking be inoculated on the test tube slant of LB substratum.
The step of purifying: by the bacterial strain inoculated in each inclined-plane, by aforementioned dilution-plate method separating step, is separated bacterial classification repeatedly, until the bacterial classification be separated is pure culture, after namely lawn grows, its colony characteristics unanimously.
4. the screening of bioflocculant-producing bacteria
A. primary dcreening operation
Each bacterial strain separation and purification gone out is inoculated in respectively in flocculation substratum and cultivates, by medium centrifugal, get the primary dcreening operation that supernatant liquor carries out flocculation activity, that is: getting 2mL nutrient solution adds in 100mL4g/L Kaolin clay suspension, contrast with the Kaolin clay suspension not adding nutrient solution simultaneously, whether observe phenomena, there is flocculation to judge whether having flocculation activity thus to carry out primary dcreening operation.If flocculation, then preliminary judgement has the ability of producing biological flocculant, needs the flocculation activity size detecting flocculation agent in nutrient solution further.Flocculation substratum is formulated as follows: NaNO
32g, KC10.5g, K
2hPO
41g, MgSO
40.5g, FeSO
40.01g, sucrose 30g and distilled water 1L, pH nature, sterilizing.
B. sieve again
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation substratum and cultivates, after 48h, using the size of flocculating rate as the size weighing bacterial strain produce flocculant ability, therefrom select the 1 strain bioflocculant-producing bacteria that flocculating effect is best.
5. the qualification of bioflocculant-producing bacteria
The bioflocculant-producing bacteria higher to this 1 strain flocculation activity filtered out carries out analysis of physio biochemical characteristics, and by Protocols in Molecular Biology, obtain the 16SrRNA gene order of each bacterial strain, then by database comparison, obtain its classified information, determine to plant belonging to it as subtilis.
(2) substratum preparation: NaNO
32g, KC10.5g, K
2hPO
41g, MgSO
40.5g, FeSO
40.01g, sucrose 30g and distilled water 1L, except FeSO
4outward, all the other medicines all at 121 DEG C of sterilizing 20min, wait gone out bacterium cooling after, by FeSO
4be dissolved in distilled water after sterilizing.In, the FeSO dissolved is filtered with sterilising filter
4, draw the FeSO after filtering with Sterile pipette
4solution adds in this substratum.
(3) ferment
This laboratory institute separation screening subtilis is out seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, under 35 DEG C of conditions after shaking culture 48h, inoculate the Erlenmeyer flask to the 250mL that 100mL substratum is housed, then shaking culture 24h under 35 DEG C of conditions.
(4) extraction of biological flocculant
Fermented liquid is put into the whizzer that rotating speed is 8000r/min, centrifugal 10min, collect supernatant liquor.Then in supernatant liquor, add the pre-cooled ethanol (4 DEG C) of 3 times of volumes, gained mixture leaves standstill 6h at 4 DEG C.Mixture is put into the whizzer that rotating speed is 10000r/min, centrifugal 10min, collecting precipitation.Dissolve 3h with appropriate distilled water, mixture is put into the whizzer that rotating speed is 8000r/min, centrifugal 10min, gained throw out dissolves 3h with distilled water again, repeatedly dialyse 1 time, to nose can't smell ethanol taste, to remove small molecules and residual organic solvent.Finally centrifugal throw out is placed in vacuum freeze drier lyophilize, biological flocculant finished product can be obtained.
(5) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
0.4 ~ 1.0g biological flocculant is added in the beaker that 100mL leather-making waste water is housed, then beaker is placed in magnetic stirring apparatus to stir, first stirs 2min with the speed of 200r/min, then stir 10min with the speed of 50r/min, filter after leaving standstill 20min, measure wherein total values of nitrogen might.
When total values of nitrogen might initial in leather-making waste water is 450mg/L, this biological flocculant can remove wherein total nitrogen 365.9mg/L, and removal efficiency reaches 81.2%, does not have secondary pollution.
Embodiment 2
(1) screening of subtilis
1. the enrichment culture of bacterial classification
Active sludge tannery got is seeded to sterilizing and in the LB substratum of cooling (substratum that the present invention mentions all be through sterilizing and cooling), cultivate 48h in 30 DEG C of shaking tables.LB substratum is formulated as follows: peptone 10g, yeast extract paste 5g, NaCl10g, distilled water 1000mL, pH7.0.121 DEG C of sterilizing 20min.
2. tamed strain
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 30 DEG C of shaking culture 48h in the LB substratum of leather-making waste water (leather-making waste water 20mL, total nitrogen 2000 degree (total nitrogen adopts total nitrogen instrument to measure)).And the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step.Its objective is the high density total nitrogen in order to enable the bacterial classification of cultivation adapt to leather-making waste water.
3. the separation of bacterial classification
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain.
(2) substratum preparation: NaNO
32g, KC10.5g, K
2hPO
41g, MgSO
40.5g, FeSO
40.01g, sucrose 30g and distilled water 1L, except FeSO
4outward, all the other medicines all at 121 DEG C of sterilizing 20min, wait gone out bacterium cooling after, by FeSO
4be dissolved in distilled water after sterilizing.In, the FeSO dissolved is filtered with sterilising filter
4, draw the FeSO after filtering with Sterile pipette
4solution adds in this substratum;
(3) ferment
This laboratory institute separation screening subtilis is out seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, under 35 DEG C of conditions after shaking culture 48h, inoculate the Erlenmeyer flask to the 250mL that 100mL substratum is housed, then shaking culture 24h under 35 DEG C of conditions.
(4) extraction of biological flocculant
Fermented liquid is put into the whizzer that rotating speed is 8000r/min, centrifugal 10min, collect supernatant liquor.Then in supernatant liquor, add the pre-cooled ethanol (4 DEG C) of 3 times of volumes, gained mixture leaves standstill 6h at 4 DEG C.Mixture is put into the whizzer that rotating speed is 10000r/min, centrifugal 10min, collecting precipitation.Dissolve 3h with appropriate distilled water, mixture is put into the whizzer that rotating speed is 8000r/min, centrifugal 10min, gained throw out dissolves 3h with distilled water again, repeatedly dialyse 1 time, to nose can't smell ethanol taste, to remove small molecules and residual organic solvent.Finally centrifugal throw out is placed in vacuum freeze drier lyophilize, biological flocculant finished product can be obtained.
(5) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
0.4 ~ 1.0g biological flocculant is added in the beaker that 100mL leather-making waste water is housed, then beaker is placed in magnetic stirring apparatus to stir, first stirs 2min with the speed of 200r/min, then stir 10min with the speed of 50r/min, filter after leaving standstill 20min, measure wherein total values of nitrogen might.
When total values of nitrogen might initial in leather-making waste water is 450mg/L, this biological flocculant can remove wherein total nitrogen 348.75mg/L, and removal efficiency reaches 77.5%, and degradation speed is fast, does not have secondary pollution.
Embodiment 3
The screening of subtilis
(1) enrichment culture of bacterial classification:
Gather the active sludge of tannery and be seeded to sterilizing and in the LB substratum of cooling, the cultivation ratio of mud and substratum is that volume ratio is about 1:8,46h is cultivated in 28 DEG C of shaking tables, LB substratum is formulated as follows: peptone 8g, yeast extract paste 4g, NaCl8g, adding distil water is to 1000mL, pH6.8,115 DEG C of sterilizing 25min; Shaking speed is 100r/min.
(2) tamed strain:
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 28 DEG C of shaking culture 50h in the LB substratum of leather-making waste water, and the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step; Taming step by step, its objective is the high density total nitrogen in order to enable the bacterial classification of cultivation adapt to leather-making waste water.
Active sludge after being cultivated by step (1), is enrichment culture bacterium liquid.
3. the separation of bacterial classification:
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain.
The screening of bioflocculant-producing bacteria
(1) primary dcreening operation
Bacillus subtilis strain separation and purification gone out is inoculated in flocculation substratum and cultivates, by medium centrifugal, get the primary dcreening operation that supernatant liquor carries out flocculation activity, that is: getting 2mL nutrient solution adds in 100mL4g/L Kaolin clay suspension, contrast with the Kaolin clay suspension not adding nutrient solution simultaneously, observe, whether there is flocculation to judge whether there is flocculation activity thus to carry out primary dcreening operation, if flocculation, then preliminary judgement has the ability of producing biological flocculant, needs the flocculation activity size detecting flocculation agent in nutrient solution further; Flocculation substratum is formulated as follows: NaNO
31g, KC10g, K
2hPO
40.5g, MgSO
40.3g, FeSO
40.01g, sucrose 27g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115 DEG C of sterilizing 25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter, then by the FeSO after filtration
4solution adds in this substratum, flocculation medium pH 6.7;
(2) multiple sieve
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation substratum and cultivates, after 45-50h, using the size of flocculating rate as the size weighing bacterial strain produce flocculant ability, therefrom select the subtilis that there is biological flocculant and produce.The subtilis filtered out can produce flocculant, has the ability removing pollutent.
The method of subtilis process leather-making waste water total nitrogen is utilized to comprise the following steps:
L () substratum is prepared: NaNO
31.5g, KC10g, K
2hPO
40.6g, MgSO
40.3g, FeSO
40.01g, sucrose 28g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115 DEG C of sterilizing 25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter the FeSO dissolved with sterilising filter
4, draw the FeSO after filtering with Sterile pipette
4solution adds in this substratum, medium pH 6.7;
(2) ferment: Bacillus subtilis strain is seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, shaking culture 45h under 32-38 DEG C of condition; Bacterial classification after cultivation inoculates the 250mL Erlenmeyer flask to being equipped with 100mL substratum, and then under 32 DEG C of conditions, shaking culture 24h obtains fermented liquid, and the inoculum size ratio of bacterial classification and substratum is volume ratio 1:8; Fermented liquid refers to that step (2) is through cultivating the nutrient solution of 24h.
(3) extraction of biological flocculant: fermented liquid is put into the whizzer that rotating speed is 10000r/min, centrifugal 10min, collect supernatant liquor, then in supernatant liquor, add the pre-cooled ethanol of 4 times of volumes, temperature 4 DEG C, mixture is put into the whizzer that rotating speed is 10000r/min leave standstill 5h at 5 DEG C after by gained mixture again, centrifugal 12min, collecting precipitation, 3h is dissolved with distilled water, lysate is put into the whizzer that rotating speed is 10000r/min, centrifugal 12min, gained throw out dissolves 3h with distilled water again, repeatedly dialyse 2 times, to nose can't smell ethanol taste, to remove small molecules and residual organic solvent, finally centrifugal throw out is placed in vacuum freeze drier lyophilize, biological flocculant finished product can be obtained,
(4) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
0.4 ~ 1.0g biological flocculant is added in the beaker that 100mL leather-making waste water is housed, then beaker is placed in magnetic stirring apparatus to stir, first stirs 2min with the speed of 200r/min, then stir 10min with the speed of 50r/min, filter after leaving standstill 20min, measure wherein total values of nitrogen might.
When total values of nitrogen might initial in leather-making waste water is 580mg/L, this biological flocculant can remove wherein total nitrogen 436.74mg/L, and removal efficiency reaches 75.3%, and degradation speed is fast, does not have secondary pollution.
Embodiment 4
The screening of subtilis
(1) enrichment culture of bacterial classification:
Gather the active sludge of tannery and be seeded to sterilizing and in the LB substratum of cooling, the cultivation ratio of mud and substratum is that volume ratio is about 1:12,46h is cultivated in 32 DEG C of shaking tables, LB substratum is formulated as follows: peptone 12g, yeast extract paste 7g, NaC12g, adding distil water is to 1000mL, pH7.2,125 DEG C of sterilizing 25min; Shaking speed is 100r/min.
(2) tamed strain:
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 28-32 DEG C of shaking culture 45-50h in the LB substratum of leather-making waste water, and the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step; Taming step by step, its objective is the high density total nitrogen in order to enable the bacterial classification of cultivation adapt to leather-making waste water.
Active sludge after being cultivated by step (1), is enrichment culture bacterium liquid.
3. the separation of bacterial classification:
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain.
The screening of bioflocculant-producing bacteria
(1) primary dcreening operation
Bacillus subtilis strain separation and purification gone out is inoculated in flocculation substratum and cultivates, by medium centrifugal, get the primary dcreening operation that supernatant liquor carries out flocculation activity, that is: getting 2mL nutrient solution adds in 100mL4g/L Kaolin clay suspension, contrast with the Kaolin clay suspension not adding nutrient solution simultaneously, observe, whether there is flocculation to judge whether there is flocculation activity thus to carry out primary dcreening operation, if flocculation, then preliminary judgement has the ability of producing biological flocculant, needs the flocculation activity size detecting flocculation agent in nutrient solution further; Flocculation substratum is formulated as follows: NaNO
33g, KC11g, K
2hPO
41.5g, MgSO
40.7g, FeSO
40.01g, sucrose 32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 125 DEG C of sterilizing 25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter, then by the FeSO after filtration
4solution adds in this substratum, flocculation medium pH 7.2;
(2) multiple sieve
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation substratum and cultivates, after 50h, using the size of flocculating rate as the size weighing bacterial strain produce flocculant ability, therefrom select the subtilis that there is biological flocculant and produce.The subtilis filtered out can produce flocculant, has the ability removing pollutent.
The method of subtilis process leather-making waste water total nitrogen is utilized to comprise the following steps:
L () substratum is prepared: NaNO
32.5g, KC11g, K
2hPO
41.2g, MgSO
40.7g, FeSO
40.01g, sucrose 32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 125 DEG C of sterilizing 25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter the FeSO dissolved with sterilising filter
4, draw the FeSO after filtering with Sterile pipette
4solution adds in this substratum, medium pH 7.2;
(2) ferment: Bacillus subtilis strain is seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, shaking culture 50h under 38 DEG C of conditions; Bacterial classification after cultivation inoculates the 250mL Erlenmeyer flask to being equipped with 100mL substratum, and then under 38 DEG C of conditions, shaking culture 68h obtains fermented liquid, and the inoculum size ratio of bacterial classification and substratum is volume ratio 1:12; Fermented liquid refers to that step (2) is through cultivating the nutrient solution of 68h.
(3) extraction of biological flocculant: fermented liquid is put into the whizzer that rotating speed is 5000r/min, centrifugal 10min, collect supernatant liquor, then in supernatant liquor, add the pre-cooled ethanol of 2 times of volumes, temperature 4 DEG C, mixture is put into the whizzer that rotating speed is 5000r/min leave standstill 5-7h at 3 DEG C after by gained mixture again, centrifugal 8min, collecting precipitation, 3h is dissolved with distilled water, lysate is put into the whizzer that rotating speed is 5000r/min, centrifugal 8min, gained throw out dissolves 2h with distilled water again, repeatedly dialyse 1 time, to nose can't smell ethanol taste, to remove small molecules and residual organic solvent, finally centrifugal throw out is placed in vacuum freeze drier lyophilize, biological flocculant finished product can be obtained,
(4) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
0.4 ~ 1.0g biological flocculant is added in the beaker that 100mL leather-making waste water is housed, then beaker is placed in magnetic stirring apparatus to stir, first stirs 2min with the speed of 200r/min, then stir 10min with the speed of 50r/min, filter after leaving standstill 20min, measure wherein total values of nitrogen might.
When total values of nitrogen might initial in leather-making waste water is 400mg/L, this biological flocculant can remove wherein total nitrogen 330.4mg/L, and removal efficiency reaches 82.6%, and degradation speed is fast, does not have secondary pollution.
The present invention is not limited to aforesaid embodiment.The present invention expands to any new feature of disclosing in this manual or any combination newly, and the step of the arbitrary new method disclosed or process or any combination newly.
Claims (8)
1. a subtilis (
bacillussubtilis) application, it is characterized in that: its preserving number of described subtilis is CCTCCM2014512, be applied to and reduce total nitrogen in leather-making waste water.
2. according to the application of a kind of subtilis described in claim 1, it is characterized in that: described subtilis is by cultivating in the active sludge of tannery and taming and obtain.
3., according to the application of a kind of subtilis described in claim 1 or 2, it is characterized in that: described cultivation and domestication step as follows: the enrichment culture of (1) bacterial classification:
Gather the active sludge of tannery and be seeded to sterilizing and in the LB substratum of cooling, the cultivation ratio of mud and substratum is that volume ratio is about 1:8-12,46-50h is cultivated in 28-32 DEG C of shaking table, LB substratum is formulated as follows: peptone 8-12g, yeast extract paste 4-7g, NaCl8-12g, adding distil water is to 1000mL, pH6.8-7.2,115-125 DEG C of sterilizing 15-25min;
(2) tamed strain:
5mL enrichment culture bacterium liquid is inoculated in 100mL and contains 28-32 DEG C of shaking culture 45-50h in the LB substratum of leather-making waste water, and the amount increasing leather-making waste water is gradually to 40mL, 60mL, 80mL, tames step by step;
(3) separation of bacterial classification:
By dilution-plate method, take solid LB media as isolation medium, from the bacterium liquid of having tamed, isolate various bacterium, and purifying is carried out to each bacterial strain, obtain Bacillus subtilis strain.
4. according to the application of a kind of subtilis described in claim 3, it is characterized in that: described cultivation and domestication also comprise the screening of bioflocculant-producing bacteria, as following steps:
(1) primary dcreening operation
Bacillus subtilis strain separation and purification gone out is inoculated in flocculation substratum and cultivates, by medium centrifugal, get the primary dcreening operation that supernatant liquor carries out flocculation activity, that is: getting 2mL nutrient solution adds in 100mL4g/L Kaolin clay suspension, contrast with the Kaolin clay suspension not adding nutrient solution simultaneously, observe, whether to occur flocculation to judge whether there is flocculation activity thus to carry out primary dcreening operation, if flocculation, then preliminary judgement has the ability of producing biological flocculant; Flocculation substratum is formulated as follows: NaNO
31-3g, KC10-11g, K
2hPO
40.5-1.5g, MgSO
40.3-0.7g, FeSO
40.01g, sucrose 27-32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115-125 DEG C of sterilizing 15-25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter, then by the FeSO after filtration
4solution adds in this substratum;
(2) multiple sieve
The bacterial strain with flocculation ability just sifted out is inoculated in respectively in flocculation substratum and cultivates, after 45-50h, using the size of flocculating rate as the size weighing bacterial strain produce flocculant ability, therefrom select the subtilis that there is biological flocculant and produce.
5. the application of a kind of subtilis according to any one of claim 1-4, is characterized in that: utilize spindle gemma
The method of bacillus process leather-making waste water total nitrogen comprises the following steps:
L () substratum is prepared: NaNO
31.5-2.5g, KC10-11g, K
2hPO
40.6-1.2g, MgSO
40.3-0.7g, FeSO
40.01g, sucrose 28-32g and distilled water 1L, except FeSO
4outward, all the other raw materials all at 115-125 DEG C of sterilizing 15-25min, wait gone out bacterium cooling after, by FeSO
4be dissolved in the distilled water after sterilizing, filter, then by the FeSO after filtration
4solution adds in this substratum;
(2) ferment: Bacillus subtilis strain is seeded to the 250mL shaking flask that 50mL substratum is housed, be placed in shaking table that rotating speed is 180r/min, shaking culture 45-50h under 32-38 DEG C of condition; Bacterial classification after cultivation inoculates the 250mL Erlenmeyer flask to being equipped with 100mL substratum, and then under 32-38 DEG C of condition, shaking culture 24 ~ 68h obtains fermented liquid, and the inoculum size ratio of bacterial classification and substratum is volume ratio 1:8-12;
(3) extraction of biological flocculant: fermented liquid is put into the whizzer that rotating speed is 5000-10000r/min, centrifugal 10min, collect supernatant liquor, then in supernatant liquor, add the ethanol of 2-4 times of volume, mixture is put into the whizzer that rotating speed is 5000 ~ 10000r/min leave standstill 5-7h at 3-5 DEG C after by gained mixture again, centrifugal 8-12min, collecting precipitation, 2.5-3.5h is dissolved with distilled water, lysate is put into the whizzer that rotating speed is 5000 ~ 10000r/min, centrifugal 8-12min, gained throw out dissolves 2-3h with distilled water again, repeatedly dialyse 1 ~ 2 time, again centrifugal throw out is placed in vacuum freeze drier lyophilize, obtain biological flocculant finished product,
(4) wastewater treatment: biological flocculant is added and is equipped with in the beaker of leather-making waste water, react on magnetic stirring apparatus.
6. according to the application of a kind of subtilis described in claim 4 or 5, it is characterized in that: the pH6.7-7.2 of described flocculation substratum or substratum.
7. according to the application of a kind of subtilis described in claim 5, it is characterized in that: in described step (3), ethanol is pre-cooled ethanol, temperature 3-5 DEG C.
8. according to the application of a kind of subtilis described in claim 5, it is characterized in that: in described step (3), the rotating speed of whizzer is 8000r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510090592.5A CN105087419B (en) | 2015-02-28 | 2015-02-28 | A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510090592.5A CN105087419B (en) | 2015-02-28 | 2015-02-28 | A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105087419A true CN105087419A (en) | 2015-11-25 |
| CN105087419B CN105087419B (en) | 2018-07-20 |
Family
ID=54568755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510090592.5A Active CN105087419B (en) | 2015-02-28 | 2015-02-28 | A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087419B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108947679A (en) * | 2018-08-17 | 2018-12-07 | 江西省峯野生物科技有限公司 | A kind of microbial organic fertilizer and preparation method thereof |
| CN109722450A (en) * | 2019-03-19 | 2019-05-07 | 西南科技大学 | Bacillus subtilis induces the method for preparing high specific surface area porous hydroxyapatite |
| CN112877247A (en) * | 2021-02-24 | 2021-06-01 | 华南理工大学 | Method for treating high ammonia nitrogen wastewater by using bacillus |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040219651A1 (en) * | 2003-01-22 | 2004-11-04 | Heng-Chuan Wang | Novel biological floculants and production methods |
| CN102337299A (en) * | 2011-10-25 | 2012-02-01 | 杭州江南科学研究院有限公司 | Preparation method of bacillus flocculant |
| CN102628066A (en) * | 2012-04-17 | 2012-08-08 | 山西大学 | Preparation method and application of microbial flocculant |
| CN102925489A (en) * | 2012-10-10 | 2013-02-13 | 临安天川环保科技有限公司 | Preparation method of microbial flocculant |
| CN103214101A (en) * | 2012-01-19 | 2013-07-24 | 中国中化股份有限公司 | Microbial flocculating agent, and preparation and use thereof |
| CN103787548A (en) * | 2014-01-26 | 2014-05-14 | 河南迪诺环保科技股份有限公司 | Biological tannery wastewater treatment system and treatment method thereof |
-
2015
- 2015-02-28 CN CN201510090592.5A patent/CN105087419B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040219651A1 (en) * | 2003-01-22 | 2004-11-04 | Heng-Chuan Wang | Novel biological floculants and production methods |
| CN102337299A (en) * | 2011-10-25 | 2012-02-01 | 杭州江南科学研究院有限公司 | Preparation method of bacillus flocculant |
| CN103214101A (en) * | 2012-01-19 | 2013-07-24 | 中国中化股份有限公司 | Microbial flocculating agent, and preparation and use thereof |
| CN102628066A (en) * | 2012-04-17 | 2012-08-08 | 山西大学 | Preparation method and application of microbial flocculant |
| CN102925489A (en) * | 2012-10-10 | 2013-02-13 | 临安天川环保科技有限公司 | Preparation method of microbial flocculant |
| CN103787548A (en) * | 2014-01-26 | 2014-05-14 | 河南迪诺环保科技股份有限公司 | Biological tannery wastewater treatment system and treatment method thereof |
Non-Patent Citations (6)
| Title |
|---|
| CHANGQING ZHAO等: "Preparation of microbial flocculant and its preliminary application on removal of ammonia in tannery effluent", 《ADVANCED MATERIALS RESEARCH VOLS》 * |
| HARUHIKO YOKOI等: "Characteristics of a Biopolymer Flocculant Produced by Bacillus sp. PY-90", 《JOURNAL OF FERMENTATION AND BIOENGINEERING》 * |
| 夏四清等: "枯草芽孢杆菌产高效微生物絮凝剂的研究", 《中国给水排水》 * |
| 杨延梅等: "微生物絮凝剂产生菌的筛选及其培养条件研究", 《重庆交通学院学报》 * |
| 董双石等: "微生物絮凝剂处理废水的研究和发展趋势", 《生物技术》 * |
| 黄凯等: "微生物絮凝剂产生菌的筛选及其培养条件研究", 《环境科学与技术》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108947679A (en) * | 2018-08-17 | 2018-12-07 | 江西省峯野生物科技有限公司 | A kind of microbial organic fertilizer and preparation method thereof |
| CN109722450A (en) * | 2019-03-19 | 2019-05-07 | 西南科技大学 | Bacillus subtilis induces the method for preparing high specific surface area porous hydroxyapatite |
| CN112877247A (en) * | 2021-02-24 | 2021-06-01 | 华南理工大学 | Method for treating high ammonia nitrogen wastewater by using bacillus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105087419B (en) | 2018-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104628225B (en) | A kind of processing method containing ammonia nitrogen industrial wastewater | |
| CN105132302A (en) | Application of bacillus cereus in processing of tannery wastewater COD | |
| JP6346982B1 (en) | Method for isolating Raulterra microorganisms, method for producing plant waste treatment agent, and method for treating plant waste | |
| CN106754481B (en) | Method for screening high-salt-resistant nitrogen-fixing strain from iceberg and related culture medium | |
| CN103589672A (en) | Method for preparing effective microorganism water quality purification agent | |
| CN117603888B (en) | Bacillus cereus and application thereof in cultivation tail water treatment | |
| CN114988965A (en) | Sludge and derivative with water content of eighty percent treated at high speed and preparation method thereof | |
| CN105087419A (en) | Application of bacillus subtilis in treating total nitrogen in tannery wastewater | |
| CN106591169B (en) | A kind of N-Methyl pyrrolidone degradation bacillus NMP-2 and its application | |
| CN113354100A (en) | Method for recycling nitrogen and phosphorus resources and neutralizing carbon by coupling filamentous algae after moderately nitrifying high-concentration ammonia nitrogen wastewater | |
| CN106865804B (en) | Clean production method of Zhongshengmycin mother medicine | |
| CN111057656B (en) | Yeast for efficiently degrading waste liquid in ice cream production and application thereof | |
| CN109609412B (en) | Thermophilic bacterium Bacillus smithii Ths1 and application thereof | |
| CN115491338B (en) | Chemical intermediate degrading strain and application thereof in wastewater treatment | |
| CN109266573B (en) | Fermentation agent and microbial inoculum for treating landfill leachate and reverse osmosis concentrated solution and treatment method | |
| CN108977365B (en) | Penicillium oxalicum L5 and application thereof in degradation of lincomycin bacterial residues | |
| CN105586331A (en) | Method of denitrified composite microbial inoculant | |
| CN116286523A (en) | Bacillus subtilis and application thereof | |
| CN114015611B (en) | Microbial composition and method for treating wastewater by using same | |
| CN112746040A (en) | Organic garbage biodegradation strain | |
| CN110803947A (en) | Manufacturing method for producing efficient water flush fertilizer by utilizing biogas slurry | |
| KR102200101B1 (en) | Composition for removing an ammonia and a bad smell and method for removing an ammonia and a bad smell using the same | |
| CN112661370A (en) | Method for treating sludge by using organisms | |
| CN105132303B (en) | A kind of application of bacillus fusiformis in handling leather-making waste water coloration | |
| CN110078305A (en) | A kind of breeding waste comprehensive utilization prevention and treatment pollution of area source method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |