CN109956562A - For handling the preparation of the Rhodococcus sp and its biological flocculant of kelp processing waste water - Google Patents
For handling the preparation of the Rhodococcus sp and its biological flocculant of kelp processing waste water Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F1/00—Treatment of water, waste water, or sewage
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Abstract
The invention discloses one plant for handling the Rhodococcus sp strain of kelp processing waste water and the preparation of biological flocculant, the Rhodococcus spRhodococcusSp SC-3 has been preserved in China General Microbiological culture presevation administrative center, deposit number at present are as follows: CGMCC NO.13268.Qualitative Identification is carried out to the produced flocculant of the bacterial strain, it was demonstrated that the produced flocculant of the bacterial strain is glycoprotein, and fermentation liquid is the biological flocculant, can be used directly after simple process or is used cooperatively with flocculation aid, for handling kelp processing waste water.Compared with prior art, the flocculation efficiency of object flocculant produced by the bacterial strain reaches 85%, and preparation method is simple, effect is good, it is low in cost, do not generate any environmental pollution, for microbial method produce flocculant carried out beneficial exploration.
Description
Technical field
The invention belongs to microorganism ambits, and in particular to for handling the Rhodococcus sp and its biology of kelp processing waste water
The preparation of flocculant.
Background technique
Kelp is that China cultivates largest algae culturing kind, and yield occupies first place in the world, and China produces kelp per year up to 148
Ten thousand tons.Currently, the processing of kelp product mainly includes dry kelp, salt marsh kelp, algin production, the life of kelp iodine, mannitol
Produce etc..A large amount of waste water can be discharged during kelp processing, major pollutants are suspended matter (SS) and a small amount of gold in waste water
Belong to ion, acid and organic matter etc., COD (COD) and biochemical oxygen demand (BOD) (BOD) are relatively low.Therefore, processing kelp adds
The suspended matter of work waste water focused in processing waste water.
The method of these suspended matters of conventional process is mainly flocculence, i.e., by adding certain ratio into waste water to be processed
The flocculant of example, makes suspended matter or colloidal substance not easily settled in water body get rid of stable state, mutual adsorption coagulation, in gravity
Effect is lower to be settled, and keeps supernatant limpid, to realize separation of solid and liquid.Currently, in the market common flocculant mainly have with
Alum, aluminium polychloride and poly-ferric chloride are the inorganic flocculating agent of representative and are the organic of representative with polyacrylamide (PAM)
Flocculant.Inorganic flocculating agent have many advantages, such as using pH range is wide, alumen ustum greatly and again, the rate of settling it is fast and cheap.But
It is that inorganic flocculating agent contains a large amount of metal ions, metal ion residual is had in the water body being processed, these metal ions are such as
Fruit, with regard to direct emission, may finally be enriched to human body by food chain without secondary treatment, and it is strong to influence human body
Health.Though organic flocculant is active by force, dosage is low, using the advantages that pH range is wide, the rate of settling is fast.But PAM monomer has
The toxicity such as carcinogenic, teratogenesis can generate if PAM is emitted into environment without processing under the degradation of environmental microorganism
Toxic monomers endanger human health, in addition, polyacrylamide production difficulty is big, risk is high, and manufacturer, China is less, and
Small scale leads to PAM heavy dependence import, at high price.
Microbial flocculant (Microbial Flocculant, abbreviation MBF) is that by micro-organisms there is flocculation to live
Property Extracellular metabolism, predominantly high-molecular compounds such as glycoprotein, mucopolysaccharide, protein, cellulose have nontoxic, easy
Degradation, flocculation range be wide, the advantages that flocculation efficiency is high, without secondary pollution, due to the advantage of MBF can overcome inorganic flocculating agent and
The inherent shortcoming of organic flocculant, and then become the research hotspot of sewage treatment in recent years.
Summary of the invention
It is an object of the invention to solve deficiency in the prior art, one plant is provided for handling the red of kelp processing waste water
Coccus SC-3, fermentation liquid can be used as biological flocculant for handling kelp processing waste water, for microbial method produce flocculant into
Beneficial exploration is gone.
The technical solution of the present invention is as follows: Rhodococcus sp new strains disclosed by the inventionRhodococcusSp. SC-3 in
On November 14th, 2016 is deposited in China Microbial Culture Preservation Commission's common micro-organisms center, deposit number CGMCC
NO.13268。
The Rhodococcus sp new strainsRhodococcusSp. SC-3 is isolated from the dirt of Jiangsu Jin Maoyuan biochemical industry Co., Ltd
Activated sludge in the aerobic tank and sedimentation basin of water treatment plant can be grown, suitable growth temperature in the environment that pH value is 6.0
It is 30 DEG C.
For handling the Rhodococcus sp new strains of kelp processing waste waterRhodococcusSp. the tool of SC-3 biological flocculant
Preparation step are as follows:
1) preparation of actication of culture and seed liquor: by the bacterial strain of cryo-conservationRhodococcusSp. SC-3 is with 4% inoculum concentration
It is inoculated in minimal medium, culture solution is cultivated 48 hours in 30 DEG C of shaking table culture case, until OD600Reach 2.0, obtains
To fermentation liquid；
The formula of minimal medium are as follows: 10 g of glucose, water 1 L, K2HPO46.70 g, KH2PO41.70 g, NH4(SO4)2
2.70 g, 10 mL of salting liquid, 108 DEG C, sterilize 20 min,
Salting liquid formula: MgSO4 19.5 g/L, MnSO45 g/L, MnSO4·H2O 5.6 g/L, FeSO45g/L, FeSO4·
7H2O 9.15 g/L, CaCl20.3 g/L；
2) preparation of flocculant: by fermentation liquid under conditions of 5000 r/min under be centrifuged 3-5 min, stay supernatant, 3 times of bodies be added
Product ethyl alcohol, 4 DEG C of refrigerators stand 24 hours, outwell supernatant collection sediment, and remaining ethyl alcohol is vapored away in 50 DEG C of baking ovens, is done
It is dry to obtain flocculant crude product, crude product flocculant is dissolved in distilled water, is configured to slightly mention flocculant.
The beneficial effects of the present invention are:
The present invention is using the Rhodococcus sp SC-3 that separates from environment as starting strain, after being activated in seed culture medium,
It is inoculated in best medium and ferments, and then the flocculant yield prepared can reach higher level.The flocculation of the biological flocculant
Effect is good and preparation method is simple, being capable of artificial large-scale culture, it is easy to accomplish industrialized production has good development and application
Prospect has carried out beneficial exploration for microbial method fermenting and producing flocculant.
Detailed description of the invention
Fig. 1 is Rhodococcus spRhodococcusSp. the electromicroscopic photograph of SC-3；
Fig. 2 is Rhodococcus spRhodococcusSp. the ultraviolet-visible spectrogram of the produced flocculant of SC-3；
Fig. 3 is Rhodococcus spRhodococcusSp. the infrared visible light spectrogram of the produced flocculant of SC-3；
Fig. 4 is Rhodococcus spRhodococcusSp. the phylogenetic tree map of SC-3；
Fig. 5 is Rhodococcus spRhodococcusSp. SC-3 handles the effect statistical form of kelp processing waste water, wherein preceding 1 indicates
The front of sewage natural sedimentation basin, rear 1 indicates the rear portion of sewage natural precipitating biochemistry pool.It is preceding 2 indicate flocculant to preceding 1 waste water at
The turbidity of supernatant after reason；2 indicate flocculant to the turbidity of supernatant after rear 1 wastewater treatment afterwards.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where essence of the present invention, to modification made by the method for the present invention, step or condition and replaces, belong to model of the invention
It encloses.
1: one plant of the embodiment Rhodococcus sp new strains for kelp processing wastewater treatmentRhodococcusSp. SC-3 has been
Through being deposited in China Microbial Culture Preservation Commission's common micro-organisms center on November 14th, 2016, preservation address is Beijing
The institute 3 of city, North Star West Road, Chaoyang District 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC NO.13268.
Screening, separation and the identification of Rhodococcus sp SC-3 bacterial strain
The screening of purpose bacterial strain: it will be taken from the aerobic tank and sedimentation basin in sewage treatment plant, Jin Maoyuan biochemical industry Co., Ltd
The activated sludge wastewater concussion come, is uniformly mixed it, carries out gradient dilution later: therefrom drawing 1 mL with liquid-transfering gun first,
A test tube is injected, is denoted as 100, then takes 1 mL to inject second test tube, and 9 mL distilled water are added, is denoted as 10-1；To 10-1Concussion
It shakes up and 1 mL is therefrom taken to inject third test tube, and add 9 mL distilled water, be denoted as 10-2；And so on, obtain 10-3, 10-4, 10-5With 10-6The dilute solution of gradient.Respectively taking 500 μ L to be coated in primary dcreening operation culture medium from the dilution of the above various concentration (can
20 g of soluble starch, agar 20 g, KNO31 g, NaCl 0.5 g, K2HPO4 0.5 g, MgSO40.5 g, FeSO4 0.01
G, water 1L, pH 7.0-7.2) on, 24 h are cultivated in 30 DEG C of insulating boxs.
Plate streaking distinguishes different bacterium colonies according to the difference of colonial morphology, color after occurring single bacterium colony on plate,
Different bacterium colonies are picked them separately with oese to cross on culture medium flat plate culture.
Different strains after scribing line culture are seeded in sterilizing minimal medium (100/250ml), 30 DEG C, 160 r/
Min cultivates 72 h.To 2 mL fermentation liquids of addition and 3 mL CaCl in aqueous suspension ofkaolin (100 mL)2(w/v, 1%) adjusts pH
Value observes suspension sedimentation effect, retains the bacterial strain that aqueous suspension ofkaolin can be made to precipitate to 7.0, hand 5 seconds.
The identification of bacterial strain Physiology and biochemistry is carried out referring to " primary Jie Shi Bacteria Identification handbook "；The DNA of new strains SC-3 is extracted, to go out
The genomic DNA of bacterium germination strain is template, is expanded using the universal primer of bacterial 16 S rDNA
Primer 1:5 '-AGAGTTTGATCCTGGCTCAG-3 '
Primer 2: 5 '-GGTTACCTTGTTACGACTT-3 '
PCR method is as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of denaturation 50 s, 58 DEG C of annealing 1.5 min, 72 DEG C of extension 2 min, 35
A circulation；72 DEG C of 10 min of extension.By 0.8% agarose gel electrophoresis of amplified production, Goldview is dyed, in the UV lamp
Observed result, connection pMD-18T carrier converts e. coli jm109 after glue recycling E. coliAfter Amp plate screening
Shanghai bio-engineering corporation is handed over to be sequenced.Have sequence with database in NCBI and be compared analysis, uses
ClustalX1.8 carries out multiple sequence matching arrangement, and mega4.0 phylogenetic tree construction is seen from development tree result combining form
It examines and bio-chemical characteristics, determines that new strains SC-3 is Rhodococcus spRhodococcus sp.。
Embodiment 2: extraction, spectroscopy and the research of aqueous suspension ofkaolin precipitation experiments of flocculant.
1) preparation of actication of culture and seed liquor: by the bacterial strain of cryo-conservationRhodococcusSp. SC-3 connects with 4%
Kind amount is inoculated in minimal medium, culture solution is cultivated 48 hours in 30 DEG C of shaking table culture case, until OD600Reach
2.0, obtain fermentation liquid；
The formula of minimal medium are as follows: 10 g of glucose, water 1 L, K2HPO46.70 g, KH2PO41.70 g, NH4(SO4)2
2.70 g, 10 mL of salting liquid, 108 DEG C, sterilize 20 min,
Salting liquid formula: MgSO4 19.5 g/L, MnSO45 g/L, MnSO4·H2O 5.6 g/L, FeSO45g/L, FeSO4·
7H2O 9.15 g/L, CaCl20.3 g/L；
2) flocculant broth extraction purifies:
Fermentation liquid is centrifuged 3min at 5000 r/min, stays supernatant.3 times of volume ethanols are added, 4 DEG C of refrigerators stand 24 hours,
Supernatant collection sediment is outwelled, vapors away remaining ethyl alcohol in 50 DEG C of baking ovens, dry flocculant crude product.Crude product is flocculated
Agent is dissolved in 100mL distilled water (primary fermentation liquid product is identical therewith), is configured to slightly mention flocculant.Measure flocculation activity.
The ultraviolet spectra of MBF-SC-3: contain the solution of MBF-C3 sterling in 200-500 nm length scanning.
The infrared spectroscopy of MBF-SC-3: it is fully ground after the flocculant of 1-2 mg or so is mixed with dry KBr, tabletting
It is afterwards 4000~400 cm in spectral domain-1In the range of carry out flocculant infrared spectroscopy spectroscopic assay.
Characteristic absorption peak is 3438.07,1653.88,1123.60,619.65 as can be seen from Figure 2.
3438.07 cm-1For wide absorption peak, 3400-3480 is-OH absorption peak, illustrates that the flocculant contains a large amount of-OH
Base, the stretching vibration of-OH cause appearance obvious；1653.88 cm-1There are a weaker absorption peak, the absorption that 1680-1600 is
Peak is-C=C- absorption peak, it is known that contains the C-O stretching vibration in a small amount of-C=C- or saccharide ring；1123.60 cm-1For simple spike,
1275-1020 is the C-O-C key of ether.619.65 cm-1It is possible that there are two types of peaks, first is that aromatic hydrocarbon C-H(900-690, plane excurvation
It is bent), another kind is C=C(1000-650, out-of plane bending).Again because ultraviolet spectra in, be smoothed curve, no absorption peak, and
If having aromatic hydrocarbon that will show absorption peak in ultraviolet spectra, therefore the polysaccharide, without aromatic hydrocarbon, 619 peaks show there is C=C.Therefore,
MBF-C3 contains the characteristic group of polysaccharide, determines that it is anionic polysaccharides flocculant.
Determine whether MBF-SC-3 contains protein component using Bradford method.
Determine whether MBF-SC-3 contains reduced sugar ingredient using DNS method.
The measurement of MBF-SC-3 polysaccharide component: taking 20 mg samples, and 20 mL H are added2SO4Solution (v/v, 20%), in 100
6 h are heated in DEG C baking oven, adjust pH to 6.0, supernatant is retained after centrifugation, carry out HPLC measurement after crossing film.
Identify that the biological flocculant is glycoprotein, polysaccharide using HPLC, Coomassie brilliant blue, ultraviolet chromatography and infrared chromatography
It is mainly made of glucose and xylose, separately there is a small amount of arabinose, three kinds of sugared molar ratios are as follows: 5.25:1.64:0.24.Examine horse
Contain a small amount of protein component in the raw sugar of this brilliant blue reaction descriptions MBF-SC-3.And UV scanning is analyzed, spectrogram is smooth song
Line, infer MBF-SC-3 do not contain nucleic acid and protein, then then may the polysaccharide contain glycoprotein, Coomassie brilliant blue react
When polysaccharide be hydrolyzed into various monosaccharide and amino acids substance, and in UV scanning, the only compound of polysaccharide of measurement at
Point.It is analyzed by infrared scan, MBF-C3 contains the characteristic group of polysaccharide, determines that it is anionic polysaccharides flocculant.
Embodiment 3:Rhodococcus sp. SC-3 produces application of the flocculant when handling kelp processing waste water
40 mL of fermentation liquid is taken, the ethyl alcohol that three times volume is added directly takes the bioflocculation when biological flocculant sinks to bottom of bottle
3 mL of agent is directly used in 100 mL kelp processing waste water of processing, and 2 mL flocculation aid CaCl are added2Solution, hand conical flask 30 seconds,
Stand 6 min.
As a result as shown in figure 5, preceding 1OD550=0.288 and rear 1 OD550=0.155, show that the turbidity of the sewage is not too
Height, compared to conventional high concentrated organic wastewater, after kelp processing industry waste water is separated by solid-liquid separation by flood dragon machine, turbidity is greatly
Decline.From the point of view of preceding 1 data comparison with rear 1, rear 1 numerical value only has the 53.8% of preceding 1, that is to say, that by natural conditions
Biochemistry precipitating, the turbidity of kelp processing industry waste water can reduce by 46.2%, and aerator is simultaneously not used in this result, almost without at
This, just makes the turbidity of waste water handle nearly half.It is set thus, it can be known that kelp processing industry factory will build basic sewage treatment
It applies, only a small amount of fund need to be spent to build 20 meters one long, wide 3 meters of sedimentation basin, so that it may the treatment effect of sewage is greatly improved,
Flocculant and aerobic aeration, which even need not be added, to play a multiplier effect.If desired sewage is further processed, is needed
Want a certain amount of flocculant.It is not recommended that, because measure energy consumption is big, processing cost is higher using aerator, the most seas in China
Medium-sized and small enterprises with processing industry are difficult to bear its cost.
It can be seen that flocculating effect is preferable from preceding 2 with rear 2 numerical value, the water that can greatly improve sewage is used alone in MBF
Matter；It is more lower than the flocculating rate 85.4% of front as the flocculating rate 79.4% at rear portion, reason may be by natural biochemical treatment,
Colloidal solid becomes smaller, to increase flocculated difficulty.
The MBF that uses of this test is the Rhodococcus sp flocculant of 10 days primary fermentations, at room temperature preservation 10 days, test knot
Fruit shows that the water outlet after flocculation is limpid, and a large amount of precipitating particles are arranged at bottom, and flocculating effect is preferable.
Basic principles and main features and advantage of the invention have been shown and described above.But the foregoing is merely this hairs
Bright specific embodiment, technical characteristic of the invention are not limited thereto, and any those skilled in the art is not departing from this hair
The other embodiments obtained under bright technical solution should all cover within the scope of the patent of the present invention.
Claims (3)
1. the Rhodococcus sp new strains for handling kelp processing waste waterRhodococcusSp. SC-3 is in November, 2016
It is deposited within 14th China Microbial Culture Preservation Commission's common micro-organisms center, deposit number is CGMCC NO.13268.
2. as described in claim 1 for handling the Rhodococcus sp new strains of kelp processing waste waterRhodococcus sp. SC-
3, which is characterized in that aerobic tank and sedimentation basin of the strain isolation from Jiangsu sewage treatment plant, Jin Maoyuan biochemical industry Co., Ltd
In activated sludge, can pH value be 6.0 environment in grow, suitable growth temperature be 30 DEG C.
3. as described in claim 1 for handling the Rhodococcus sp new strains of kelp processing waste waterRhodococcus sp. SC-
The preparation of 3 biological flocculants, which is characterized in that specific preparation step are as follows:
1) preparation of actication of culture and seed liquor: by the bacterial strain of cryo-conservationRhodococcusSp. SC-3 is with 4% inoculum concentration
It is inoculated in minimal medium, culture solution is cultivated 48 hours in 30 DEG C of shaking table culture case, until OD600Reach 2.0, obtains
To fermentation liquid；
The formula of minimal medium are as follows: 10 g of glucose, water 1 L, K2HPO46.70 g, KH2PO41.70 g, NH4(SO4)2
2.70 g, 10 mL of salting liquid, 108 DEG C, sterilize 20 min,
Salting liquid formula: MgSO4 19.5 g/L, MnSO45 g/L, MnSO4·H2O 5.6 g/L, FeSO45g/L, FeSO4·
7H2O 9.15 g/L, CaCl20.3 g/L；
2) preparation of flocculant: by fermentation liquid under conditions of 5000 r/min under be centrifuged 3-5 min, stay supernatant, 3 times of bodies be added
Product ethyl alcohol, 4 DEG C of refrigerators stand 24 hours, outwell supernatant collection sediment, and remaining ethyl alcohol is vapored away in 50 DEG C of baking ovens, is done
It is dry to obtain flocculant crude product, crude product flocculant is dissolved in distilled water, is configured to slightly mention flocculant.
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| CN111548971A (en) * | 2020-06-02 | 2020-08-18 | 江苏海洋大学 | Preparation method of economic microalgae high-efficiency culture medium based on laver processing wastewater |
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|---|---|---|---|---|
| CN111548971A (en) * | 2020-06-02 | 2020-08-18 | 江苏海洋大学 | Preparation method of economic microalgae high-efficiency culture medium based on laver processing wastewater |
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Application publication date: 20190702 |