CN103602620B - A kind of bacterial strain and application thereof with high flocculation ability - Google Patents
A kind of bacterial strain and application thereof with high flocculation ability Download PDFInfo
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Abstract
The present invention relates to a kind of bacterial strain and application thereof with high flocculation ability. With the bacterial strain wall bacillus with high flocculation ability provided by the invention (<i>Bacillus? muralis</i>) metabolite of D6 is as main active, as microbial flocculant, is applied to the slaughtering wastewater of degrading, have nontoxic, can natural degradation, the feature such as good, the non-secondary pollution for the treatment of effect. Under the best adds combination condition, the COD of the microbial flocculant that bacterial strain D6 produces through fermentation to slaughtering wastewaterCr, SS, turbidity, TN, NH4 +-N clearance is respectively 92.19%, 93.06%, 97.23%, 35.91%, 38.82%, has improved wastewater treatment efficiency, for the efficient processing of slaughtering wastewater provides new method.
Description
Technical field
The present invention relates to a kind of by the sewage draining exit mud of slaughterhouse, sieve the bacterial strain with high flocculation ability, this bacterial strain warpThe microbial flocculant (MBF) that fermentation produces has efficient degradation to slaughtering wastewater.
A kind of bacterial strain called after with high flocculation ability that the present invention is alleged: wall bacillus (BacillusMuralis) D6, on October 7th, 2013 in the center preservation of Chinese Typical Representative culture collection, be numbered CCTCCNO:M2013461。
Background technology
As the water treatment agent early being used, chemical floc mainly comprises inorganic flocculating agent and organic polymer flocculationAgent, this class flocculant has the features such as cheap, treatment effect is obvious, therefore used in a large number in early days, but use procedureEasily cause metal ion and organic substance residues and cause secondary pollution.
Slaughtering wastewater contains the suspensions such as a large amount of watery blood, ight soil, soft flocks, fat, bone bits, internal organ, and with strongBad smell. Because slaughtering wastewater is many containing organism kinds, environment is caused to great pollution, conventional process effect is paid no attention toThink.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of bacterial strain wall bacillus with high flocculation ability(Bacillusmuralis) D6, on October 7th, 2013 in the center preservation of Chinese Typical Representative culture collection, be numberedCCTCCNO:M2013461. Hereinafter to be referred as bacterial strain D6.
Another object of the present invention is to provide a kind of microbial flocculant, using the metabolite of bacterial strain D6 as main activityComposition, is applied to the slaughtering wastewater of degrading, have nontoxic, can natural degradation, the spy such as good, the non-secondary pollution for the treatment of effectPoint.
The application of the microbial flocculant that above-mentioned bacterial strain D6 produces in degraded slaughtering wastewater, concrete grammar is as follows:
1) the wall bacillus that is CCTCCNO:M2013461 by above-mentioned deposit number (Bacillusmuralis) D6Bacterial classification access fermentation medium, at constant-temperature shaking incubator, with 30 DEG C of temperature, rotating speed is that 160r/min cultivates 72h, by zymotic fluidWith the centrifugal 30min of centrifuge 4000r/min, get supernatant, be microbial flocculant.
Described fermentation medium is: soluble starch 20g, KH2PO42g，K2HPO45g，(NH4)2SO40.2g，NaCl0.1g, urea 0.5g, yeast extract 0.5g, MgSO4·7H2O0.2g, distilled water 1000mL, initial pH7.0,121 DEG C of sterilizings20min。
2) in slaughtering wastewater, add microbial flocculant and CaCl2, regulate pH. In every 100mL slaughtering wastewater, add in a subtle wayBiological flocculant 0.5-3.5mL; 1%CaCl22-9mL, regulates pH value 6.0-12.0. Preferably, optimum flocculating condition
For: in every 100mL slaughtering wastewater, add microbial flocculant 2.5mL; 1%CaCl25.5mL; PH is 8.0.
The invention has the beneficial effects as follows: the microbial flocculant that the present invention produces bacterial strain D6, joins slaughtering wastewaterIn, the suspension in waste water, colloidal solid can be assembled and formed larger wadding flower, after leaving standstill, realize Separation of Solid and Liquid. The present invention carriesThe microbial flocculant of confession can degrade quickly and effectively organic matter in slaughtering wastewater, reduce turbidity, make water outlet as clear as crystal.COD, SS, turbidity etc. are all had to good treatment effect, reduced the load for the treatment of system, improve treatment effeciency. In the bestAdd under combination condition the COD of the microbial flocculant that bacterial strain D6 produces through fermentation to slaughtering wastewaterCr, SS, turbidity, TN,NH4 +-N clearance is respectively 92.19%, 93.06%, 97.23%, 35.91%, 38.82%, has improved wastewater treatment efficiency, for butcheringThe efficient processing of waste water provides new method.
Brief description of the drawings
Fig. 1 bacterial strain D6 growth curve chart.
The atomic force photo of Fig. 2 bacterial strain D6.
Fig. 3 bacterial strain D616SrDNA fragment amplification figure.
Fig. 4 bacterial strain D6 chadogram.
Fig. 5 bacterial strain D6 produces the distribution situation of microorganism flocculation activity.
The impact of the dosage of Fig. 6 bacterial strain flocculant that D6 produces on flocculating effect.
The impact of Fig. 7 pH value on flocculating effect.
Fig. 8 CaCl2The impact of dosage on flocculating effect.
Detailed description of the invention
Acquisition and the qualification of embodiment 1 wall bacillus (Bacillusmuralis) D6
(1) screening technique of D6
Adopt dilution spread and plate streak from Shenyang poultry processing factory slaughtering wastewater sewage draining exit bed mud and aeration tankIsolated strains in activated sludge, separation and purification obtains 55 strain bacterial strains, after primary dcreening operation, has the tunning of 15 strain bacterial strains can make heightRidge soil suspension produces flocculation phenomenon, has floc sedimentation to produce, and proves that its tunning has flocculating properties. By the 15 strain bacterium that obtain after primary dcreening operationMultiple sieve is carried out in strain, and with flocculating rate sign microbial flocculant flocculation ability, multiple sieve the results are shown in Table 1, wherein, and D6, B8, E7, E12'sFlocculating rate is all greater than 94%, with the flocculating rate maximum of D6, is 96.77%.
Table 1 sieves result again
(2) mensuration of bacterial strain D6 growth curve
Liquid Culture based formulas: 1000mL distilled water, beef extract 3g, peptone 10g, NaCl5g, 7.2,121 DEG C of pH valuesSterilizing 20min.
Concrete grammar: put into several glass marbles through sterilization treatment and prevent bacterial classification conglomeration in each triangular flask, then putEnter in shaking table and vibrate, shaking speed is 160r/min, and temperature is 30 DEG C, the extinction of measuring under 660nm wavelength every three hoursDegree, taking the time as abscissa, OD660 is that ordinate is drawn growth curve.
As shown in Figure 1, as seen from Figure 1, bacterial strain D6 is in 0th～3h adjustment period of growth for result, and bacterium number increasess slowly,3rd～15h enters growth logarithmic phase, and bacterium is counted sharp increase, and 15h enters the growth stage of stable development later, and bacterium number no longer includes violentChange.
(3) morphological feature of bacterial strain D6 and bacterial classification qualification result are as follows:
A. bacterial strain D6 monomer and colony characteristics
Bacterial strain D6 is bacillus, without pod membrane, and peritrichous, Fig. 2 is shown in by the atomic force photo of monomer D6 bacterium. Colony characteristics is yellowishThe circle of look, opaque, edge is non-wrinkled, and rat has stickiness.
B. the physio-biochemical characteristics of bacterial strain D6
Gram G, catalase, indoles, acetic acid are oxidized, gelatin liquefaction is positive; Methyl red, Starch Hydrolysis, VP, H2S, oilFat, ethanol, citrate react negative, not decomposing milk reindeer moss; Do not produce not aerogenesis of acid, do not reduce nitrate.
C. the order-checking of the 16SrDNA of bacterial strain D6
The extraction of the total DNA of bacterial strain D6: (one) strain culturing: bacterial strain D6 is inoculated in 5mL fluid nutrient medium, puts into shaking table160r/min, 30 DEG C are cultured to growth logarithmic phase; (2) microorganism collection: get 1mL culture in EP pipe with the pipette of sterilizing,Under room temperature, 8000 leave heart 5min, abandoning supernatant, and it is 8.0 ddH that precipitation is dissolved in to pH again2In O; (3) cellular lysate:The lysozyme soln of configuration 50mg/mL, then gets 6 μ L and joins in solution obtained in the previous step, acts on 2h at 37 DEG C, then distinguishesAdd the NaCl solution that 50 μ L concentration are 2mol/L, the SDS that 110 μ L concentration are 10%, the Proteinase K that 3 μ L concentration are 20mg/L,37 DEG C are spent the night. (4) extracting: bacterium liquid is on average assigned in the EP pipe of 2 1.5mL, the phenol, chloroform, the isoamyl that add equal-volume to configureAlcoholic solution (three's volume ratio is 25:24:1), mixes rear room temperature and places 5～10min, and 12000 leave heart 10min, extracting 2Inferior; (5) precipitation: add 0.6 times to the isopropyl alcohol of extract volume, mix rear room temperature and place 10min, 12000 leave the heart10min; (6) washing: the precipitation obtaining is washed 2 times with 75% ethanol, then 12000 leave heart 1min; (7) after drying,Be dissolved in the ddH of 50 μ L2In O, get 2～5 μ L and on the Ago-Gel preparing, carry out electrophoresis, after electrophoresis finishes, put into EB and dyeLook 20min.
Bacterial strain D616SrDNA amplification: reaction system is in table 2:
Table 216SrDNA amplification reaction system
| Template (μ L) | 2 |
| Primers F 1(μ L) sequence A GAGTTTGATCMTGGCTCAG | 2 |
| Primers F 2(μ L) sequence A AGGAGGTGMTCCARCC | 2 |
| dNTPS（μL） | 6 |
| Buffer solution (μ L) | 5 |
| RTag enzyme (μ L) | 1 |
| ddH2O（μL） | 32 |
Pcr amplification needs 94 DEG C of denaturation 5min, 94 DEG C of sex change 40s, 55 DEG C of annealing 50s, totally 35 circulations, 72 DEG C of extensions1.5min, 72 DEG C stop 7min. Pcr amplification product is got 10 μ L, 1% agarose gel electrophoresis and is detected. Pcr amplification product electrophoretogramSpectrum as shown in Figure 3. As shown in Figure 3, the 16SrDNA fragment electrophoretic band after pcr amplification, near 1600～1650bp, does not have non-Specific band and primer dimer, result is good.
DNA sequencing: order-checking is completed by the precious biotech firm in Dalian. Sequence is as shown in SEQIDNO:1.
D. the systematic evolution tree analysis of bacterial strain D6
Draw chadogram according to 16SrDNA sequencing result, as shown in Figure 4. Bacterial strain D6 and wall bacillus as seen from Figure 4(Bacillusmuralis) similitude of 16SrDNA sequence has reached 99.5%, identifies that this bacterial strain is wall bacillus（Bacillusmuralis）。
E. conclusion: with reference to " common bacteria system identification handbook " and " the outstanding Bacteria Identification handbook of uncle ", in conjunction with Physiology and biochemistry spyThe similitude of the 16SrDNA sequence of seeking peace is 99.5%, identifies that this bacterial strain D6 is wall bacillus (Bacillusmuralis). WallBacillus (Bacillusmuralis) D6, on October 7th, 2013 in the center preservation of Chinese Typical Representative culture collection, compileNumber be CCTCCNO:M2013461.
(4) microorganism flocculation activity distribution situation
Concrete grammar: configure respectively the kaolin suspension that 5 parts of 100mL concentration are 4%, label 1～5 adds in No. 1 solutionEnter 2.5mL fermenation raw liquid (containing cell), the CaCl that 5.5mL concentration is 1%2Solution, adjusting pH is that 8.0, No. 2～No. 4 samples will be sent outFerment stoste (containing cell) replaces with respectively supernatant, supernatant and bacterial suspension after sterilizing, and other conditions are constant, 200r/Min stirs 1min, and 50r/min stirs 3min, after standing 10min, measures respectively the absorbance of supernatant under 550nm wavelength, 5Number sample adds 2.5mL distilled water in contrast. As shown in Figure 5, as shown in Figure 5, what flocculation activity was the highest is by fermentation to resultSupernatant after culture medium is centrifugal, flocculation activity has reached 98.2%, is secondly the supernatant after high-temperature sterilization, flocculation activityBeing 95.7%, is fermenation raw liquid (containing cell) again, and flocculation activity reaches 93.3%, and what flocculation activity was minimum is bacterial suspension, waddingSolidifying activity only has 66.8%, explanation thus, and this microorganism flocculation main active is born of the same parents' extra-metabolite, and has betterHeat endurance.
Embodiment 2 degrade slaughtering wastewater optimum flocculating condition determine and application
(1) preparation of microbial flocculant: by bacterial strain D6 access be equipped with 50mL fermentation medium (1000mL distilled water, canSoluble starch 20g, KH2PO42g、K2HPO45g、(NH4)2SO40.2g, NaCl0.1g, urea 0.5g, yeast extract 0.5g,MgSO4·7H2O0.2g, initial pH7.0,121 DEG C of sterilizing 20min) 250mL triangular flask in, at constant-temperature shaking incubator to shakeSwing 30 DEG C of actuator temperatures, revolution is that 160r/min cultivates 72h. After cultivation by centrifuge 4000r/min centrifugal 30min for zymotic fluid,Get supernatant and be microbial flocculant.
(2) single factor optimum flocculating condition test:
A. the impact test of microbial flocculant dosage on flocculating effect
In 100mL slaughtering wastewater, fixing 1%CaCl2Consumption is 5mL, and pH is 7.0, relatively microbial flocculant dosageFlocculating effect during for 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, 3.5mL, result as shown in Figure 6, can by Fig. 6Know, in the time that flocculant dosage is 2.5mL, flocculating rate is the highest, has reached 92.1%.
The impact test of B.pH value on flocculating effect
The variation of pH value can directly have influence on the zeta potential in water body, and then affects the knot of biological flocculant and insoluble matterClose, therefore the size of pH value is the key factor that affects flocculating effect. In 100mL slaughtering wastewater, fixation of microbe flocculantDosage be 2.5mL, 1%CaCl2Consumption is 5mL, and relatively pH is respectively the flocculating effect of 6,7,8,9,10,11,12 o'clock, knotAs shown in Figure 7, as shown in Figure 7, in the time that pH value is 7, can reach the highest flocculating rate is 93.2% to fruit.
C.1%CaCl2The impact test of dosage on flocculating effect
In 100mL slaughtering wastewater, the dosage of fixation of microbe flocculant is 2.5mL, and pH is 7, relatively 1%CaCl2ThrowFlocculating effect when dosage is respectively 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, result as shown in Figure 8, can by Fig. 8Know, work as 1%CaCl2When dosage is 5.0mL, flocculating rate has been up to 93%.
(3) three factor optimum flocculating condition orthogonal tests:
With flocculant dosage, 1%CaCl2The factor that dosage, pH value are orthogonal test, according to L9(34) orthogonal trialThe orthogonal test that becomes Three factors-levels, orthogonal experiments and the results of analysis of variance are as table 3 and table 4.
Table 3 orthogonal experiments
Table 4 orthogonal test the results of analysis of variance
From the Sig value in table 4, pH has a significant impact the flocculating effect tool of bacterial strain D6 microbial flocculant, thisFurther show that wall micro-organisms bacillus flocculant plays a decisive role to flocculating effect in the abundant stretching, extension of flocculation system. And wad a quilt with cottonSolidifying agent dosage and 1%CaCl2Dosage does not have significant difference, illustrates that flocculation system has wider fitting to these two factorsAnswer scope. From the R value of table 3, each factor is on the descending pH that is followed successively by of the impact of flocculating effect > flocculant dosage > 1%CaCl2Dosage. Can be obtained the optimum flocculating condition of this microbial flocculant to slaughtering wastewater flocculation by the k value in table 3For: in every 100ml slaughtering wastewater, add microbial flocculant 2.5mL, 1%CaCl25.5mL，pH8.0。
(4) example
Add condition according to the optimum optimization drawing and (in every 100ml slaughtering wastewater, add microbial flocculant 2.5mL, 1%CaCl25.5mL, pH8.0), slaughtering wastewater is carried out to treatment effect test, measurement result is as table 5.
The clearance of every physical and chemical index under table 5 optimum optimization treatment conditions
Claims (6)
1. a bacterial strain with high flocculation ability, is characterized in that: called after wall bacillus (Bacillusmuralis)D6, in the center preservation of Chinese Typical Representative culture collection, is numbered CCTCCNO:M2013461, and its 16SrDNA sequence is as SEQShown in IDNO:1.
2. the bacterial strain with high flocculation ability claimed in claim 1 is in the application of degrading in slaughtering wastewater.
3. application as claimed in claim 2, is characterized in that method is as follows:
1) the wall bacillus (Bacillus that is CCTCCNO:M2013461 by deposit number claimed in claim 1Muralis) D6 bacterial classification access fermentation medium, at constant-temperature shaking incubator, with 30 DEG C of temperature, rotating speed is that 160r/min cultivates72h, by the centrifugal 30min of zymotic fluid centrifuge 4000r/min after cultivating, gets supernatant, is microbial flocculant;
2), in slaughtering wastewater, add microbial flocculant and CaCl2, regulate pH.
4. application as claimed in claim 3, is characterized in that: described fermentation medium is: soluble starch 20g,KH2PO42g，K2HPO45g，(NH4)2SO40.2g, NaCl0.1g, urea 0.5g, yeast extract 0.5g, MgSO4·7H2O0.2g, steamsHeat up in a steamer water 1000mL, initial pH7.0,121 DEG C of sterilizing 20min.
5. application as claimed in claim 3, is characterized in that: in every 100mL slaughtering wastewater, add microbial flocculant 0.5-3.5mL；1％CaCl22-9mL; PH value 6.0-12.0.
6. application as claimed in claim 5, is characterized in that: in every 100mL slaughtering wastewater, add microbial flocculant2.5mL；1％CaCl25.5mL; PH is 8.0.
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| CN104593301B (en) * | 2015-01-15 | 2017-07-11 | 华南农业大学 | One plant of wall bacillus G1 and its preparation method and application |
| CN106282069B (en) * | 2016-09-30 | 2019-02-05 | 福建省微生物研究所 | A kind of pair coccus and the application in sewage purification |
| CN106906157B (en) * | 2017-01-12 | 2020-09-08 | 常州大学 | Rhodococcus, method for producing flocculant by using same and application of rhodococcus in kelp processing wastewater |
| CN108101314A (en) * | 2018-01-10 | 2018-06-01 | 福建省致青生态环保有限公司 | A kind of comprehensive treatment Application way of livestock breeding wastewater |
| CN115786192B (en) * | 2022-11-15 | 2023-05-12 | 广西民族大学 | Bacillus paramycoides and application thereof |
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| CN102992463A (en) * | 2012-11-26 | 2013-03-27 | 洪仁作 | Slaughter wastewater flocculating agent |
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| 微生物絮凝剂的研究现状与发展趋势􂢊􀏢􀦥􁈩􄍟􀢓;曾玉彬等;《江苏环境科技》;20071231;第20卷(第2期);114-117 * |
| 高效微生物絮凝剂D6对屠宰废水的应用研究;付保荣等;《生态科学》;20100831;第29卷(第4期);307-311 * |
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