CN105132302A - Application of bacillus cereus in processing of tannery wastewater COD - Google Patents
Application of bacillus cereus in processing of tannery wastewater COD Download PDFInfo
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- CN105132302A CN105132302A CN201510090593.XA CN201510090593A CN105132302A CN 105132302 A CN105132302 A CN 105132302A CN 201510090593 A CN201510090593 A CN 201510090593A CN 105132302 A CN105132302 A CN 105132302A
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Abstract
The present invention discloses application of bacillus cereus in processing of tannery wastewater COD, the application of the bacillus cereus in processing of the tannery wastewater COD comprises the following steps: (1) medium preparation; (2) fermentation, to be more specific, the bacillus cereus is inoculated into a shake flask containing a medium for fermentation to obtain fermentation broth; (3) extraction of a bioflocculant, to be more specific, the fermentation broth is centrifuged, extracted, and then centrifuged again and vacuum freeze-dried to obtain a bioflocculant product; (4) wastewater processing, to be more specific, the bioflocculant is added into a beaker containing tannery wastewater, and the beaker is stirred on a magnetic stirrer. The new bioflocculant is used for processing of tannery wastewater COD, the removal efficiency can reach 75.6%, the unique properties of being green, environmentally friendly, free of secondary pollution, and the like of the bioflocculant can be played, a new method for the removal of tannery wastewater high concentration COD can be provided, application of the functions of the bacillus cereus can be broaden, and the bacillus cereus has strong application value.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of waxy Bacillus (
bacilluscereus) processing the application in leather-making waste water COD.
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 that leather-making waste water organic concentration is high, oxygen-consumption is large.According to data, leather-making waste water pollutes and ranked third position in light industry, becomes one of the difficult point and focus of current industrial wastewater treatment.It is estimated, China's leather-making waste water amount is large, year total water consumption be about 1.4 hundred million tons, water displacement is at about 1.2 hundred million tons; In the objectionable impurities of year discharge, COD15 more than ten thousand tons, the COD of tanning industry comprehensive wastewater is generally 3000-4000mg/L, and pollution problem becomes one of greatest problem that tanning industry development formation restricts.Thus, economic, the efficient COD Treatment process of research and development has become emphasis and the focus of leather-making waste water and even Water Pollution Control Engineering area research.
In leather-making waste water, COD treatment process is mainly physico-chemical processes, biochemical process and electrochemical process.But be still present in the process specific aim of high density leather water not strong, go COD efficiency not high, and easily cause problems such as environment second time pollutions.
Summary of the invention
In order to solve above technical problem, the invention provides a kind of waxy Bacillus (
bacilluscereus) application, described waxy Bacillus preserving number is CCTCCM2014513, be preserved in China typical culture collection center, preservation date is on October 26th, 2014, by extracting biological flocculant from waxy Bacillus bacterial classification, and with the COD in this leather wastewater treatment by bioflocculant, environmental protection, do not produce secondary pollution etc., the high concentration COD waste water produced in elimination tanning production to the pollution of environment, and builds COD process new technology in clean leather-making waste water.
Solve the application of a kind of waxy Bacillus of above technical problem, it is characterized in that: preserving number is CCTCCM2014513, described waxy Bacillus is applied to and reduces COD in leather-making waste water.
Described waxy Bacillus 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 concentration COD 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 COD clearance 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 COD clearance 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
Waxy Bacillus inoculation separation and purification gone out is cultivated in flocculation substratum, 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 waxy Bacillus that there is biological flocculant and produce.The waxy Bacillus filtered out can produce flocculant, has the ability removing pollutent.
A kind of application of waxy Bacillus in the present invention, is characterized in that: utilize the method for waxy Bacillus process leather-making waste water COD 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: by waxy Bacillus strain inoculation 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.
Waxy Bacillus in the present invention, through enrichment, domestication, has adapted to the environment containing high concentration COD 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.
The biological flocculant process waste water that the present invention produces with waxy Bacillus, has good cohesion and unique decolorizing effect, applied widely, is easy to biological degradation, can eliminates secondary pollution, safe and reliable, belongs to Green Product.
COD in the leather wastewater treatment by bioflocculant that the present invention produces with waxy Bacillus, play biological flocculant environmental protection, do not produce the special performances such as secondary pollution, open the biological flocculant New raxa removing COD in leather-making waste water, the high concentration COD waste water that produces in tanning production can be eliminated to the pollution of environment, and build COD process new technology in clean leather-making waste water, thus be that the high concentration COD removed in leather-making waste water provides novel method; In addition, also widen the application to waxy Bacillus 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 waxy Bacillus
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, COD2000 degree (COD adopts COD 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 concentration COD 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 COD clearance 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 COD clearance 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 waxy Bacillus.
(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 waxy Bacillus 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: 0.4 ~ 1.2g 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 stir 2min with the speed of 200r/min, 10min is stirred again with the speed of 50r/min, filter after leaving standstill 20min, the COD value before and after wastewater measurement process.
When COD value initial in leather-making waste water is 1200mg/L, this biological flocculant can remove wherein COD907.2mg/L, and removal efficiency reaches 75.6%, and degradation speed is fast, does not have secondary pollution.
Embodiment 2
(1) screening of waxy Bacillus
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, COD2000 degree (COD adopts COD 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 concentration COD 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 waxy Bacillus 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: 0.4 ~ 1.2g 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 stir 2min with the speed of 200r/min, 10min is stirred again with the speed of 50r/min, filter after leaving standstill 20min, the COD value before and after wastewater measurement process.
When COD value initial in leather-making waste water is 1200mg/L, this biological flocculant can remove wherein COD873.6mg/L, and removal efficiency reaches 72.8%, and degradation speed is fast, does not have secondary pollution.
Embodiment 3
The screening of waxy Bacillus
(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 concentration COD 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
Waxy Bacillus inoculation separation and purification gone out is cultivated in flocculation substratum, 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 waxy Bacillus that there is biological flocculant and produce.The waxy Bacillus filtered out can produce flocculant, has the ability removing pollutent.
The method of waxy Bacillus process leather-making waste water COD 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: by waxy Bacillus strain inoculation 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 ~ 1.2g 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, the COD value before and after wastewater measurement process.
When COD value initial in leather-making waste water is 1500mg/L, this biological flocculant can remove wherein COD1102.5mg/L, and removal efficiency reaches 73.5%, and degradation speed is fast, does not have secondary pollution.
Embodiment 4
The screening of waxy Bacillus
(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 concentration COD 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
Waxy Bacillus inoculation separation and purification gone out is cultivated in flocculation substratum, 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 waxy Bacillus that there is biological flocculant and produce.The waxy Bacillus filtered out can produce flocculant, has the ability removing pollutent.
The method of waxy Bacillus process leather-making waste water COD 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: by waxy Bacillus strain inoculation 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 ~ 1.2g 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, the COD value before and after wastewater measurement process.
When COD value initial in leather-making waste water is 1800mg/L, this biological flocculant can remove wherein COD1229.4mg/L, and removal efficiency reaches 68.3%, 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 waxy Bacillus (
bacilluscereus) application, it is characterized in that: its preserving number of described waxy Bacillus is CCTCCM2014513, be applied to and reduce COD in leather-making waste water.
2. according to the application of a kind of waxy Bacillus described in claim 1, it is characterized in that: described waxy Bacillus is by cultivating in the active sludge of tannery and taming and obtain.
3., according to the application of a kind of waxy Bacillus 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 waxy Bacillus bacterial classification.
4. according to the application of a kind of waxy Bacillus 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
Waxy Bacillus inoculation separation and purification gone out is cultivated in flocculation substratum, 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 waxy Bacillus that there is biological flocculant and produce.
5. the application of a kind of waxy Bacillus according to any one of claim 1-4, is characterized in that: utilize the method for bacillus fusiformis process leather-making waste water COD 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, then by the FeSO after filtration
4solution adds in this substratum;
(2) ferment: by waxy Bacillus strain inoculation 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 waxy Bacillus 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 waxy Bacillus 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 waxy Bacillus described in claim 5, it is characterized in that: in described step (3), the rotating speed of whizzer is 8000r/min.
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| CN107758869A (en) * | 2016-08-19 | 2018-03-06 | 辽宁惠源生物环保科技有限公司 | A kind of method that leather-making waste water is handled with microbial flocculant |
| CN106430632A (en) * | 2016-12-09 | 2017-02-22 | 三零（深圳）生物环保有限公司 | Microbial flocculant for treating petrochemical oily sewage as well as preparation and application of microbial flocculant |
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| CN108949625A (en) * | 2018-07-19 | 2018-12-07 | 中国药科大学 | A kind of degradation organic matter bacillus cereus and its application |
| CN110898998A (en) * | 2019-11-25 | 2020-03-24 | 太原理工大学 | Method for flotation of phlogopite by synergistic effect of microorganisms and dodecylamine |
| CN113957001A (en) * | 2021-08-26 | 2022-01-21 | 中国林业科学研究院林产化学工业研究所 | Bacillus for producing spore laccase and application thereof |
| CN113957001B (en) * | 2021-08-26 | 2023-11-17 | 中国林业科学研究院林产化学工业研究所 | Bacillus for producing spore laccase and application thereof |
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