CN1253553C - Ceramide bacillus and method for production of glycoprotein analog biological flocculant using same - Google Patents
Ceramide bacillus and method for production of glycoprotein analog biological flocculant using same Download PDFInfo
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- CN1253553C CN1253553C CN 200410023988 CN200410023988A CN1253553C CN 1253553 C CN1253553 C CN 1253553C CN 200410023988 CN200410023988 CN 200410023988 CN 200410023988 A CN200410023988 A CN 200410023988A CN 1253553 C CN1253553 C CN 1253553C
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- ceramide
- bacillus
- flocculant
- biological flocculant
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Abstract
The present invention relates to a mesophilous ceramide bacillus BF-QSH7, which is characterized in that the preservation unit is a preservation center in the wuhan city, and the preservation number is M. 204033. The present invention can be used for preparing glucoprotein bio-flocculant, when the glucoprotein bio-flocculant is prepared, (NH4)2SO4, K2HPO4 and MgSO4 used as fermentation media are added to starch wastewater, after high-temperature steam sterilization is carried out, seed culture media containing the mesophilous ceramide bacillus are added to carry out fermentation culture, and the temperature of the fermentation culture is from 25 DEG C to 35 DEG C; after ventilation, stir and culture are carried out for 20 to 48h, centrifugal separation is carried out, thalli are collected, and the thalli are extracted by diluted hydrochloric acids; after the extracting solutions are decompressed and concentrated, propanone or ethanol is added to carry out sedimentation, precipitate is centrifugally collected, and the crude product of the bio-flocculant is obtained by vacuum drying. Production cost can be greatly reduced by using the starch wastewater as raw materials, fermentation time is short, and extraction technologies are simple. The bio-flocculant has multiple active sites, high flocculating activity, wide flocculating objects and extensive use condition.
Description
Technical field
The present invention relates to thermophilic ceramide bacillus (Sphingobacterium thalpophilum) BF-QSH7 and from starch wastewater, prepare the method for a kind of novel glycoprotein analog biological flocculant SPB-1 with it.
Background technology
Flocculation agent is widely used in industrial circles such as sewage disposal, foodstuff production and biological fermentation, inorganicly often cause problem of environmental pollution but at present institute is widely used with the residue organic synthesis polymeric flocculant, even the mankind are had toxic actions such as carcinogenic, teratogenesis, forbidden by many countries or the use of limiting the quantity of.Therefore people sight turn to that exploitation is efficient, safety non-toxic, novel flocculant free from environmental pollution.
Advantages such as biological flocculant mainly is by the high-molecular biologic polymkeric substance with flocculation activity of microorganism secreted generation under Incubation Condition, has safety non-toxic, and is environmentally friendly.Dozens of countries such as the U.S., Japan, Britain are studied biological flocculant at present, filter out multiple bacterium for producing flocculant, and carried out a series of work in the Gene Handling of flocculating conditions, mechanism, produce flocculant, purifying, character and the application facet of flocculation agent.People such as Takeda isolate rhodococcus erythropolis (R.erythropolis) S-1 bacterial strain from soil, by the protein flocculation agent NOC-1 of its generation, it is remarkable to handle various waste water effects, but do not obtain widespread use because of production cost is high.(M.Takena，R.Kurane，J.Kiozumi，I.Nakamura，Agri.Biol.Chem.1991，55(10)：2663-2664)。The bacterial classification that is used to prepare biological flocculant at present is a carbon source with organism such as glucose, fructose, sucrose, ethanol mostly, is nitrogenous source with extractum carnis, peptone, yeast water etc., the production cost height, thus limited greatly in industrial widespread use.
Starch is widely used in industrial circles such as food, chemical industry, papermaking, medicine, weaving, can produce a large amount of high concentrated organic wastewaters in its production process, and directly discharging not only can cause serious environmental to pollute, and can cause the waste of resource.Each state is all in the effective way of seeking to handle starch wastewater, and studying more is to utilize starch wastewater to produce the unicellular of yeast, mould, bacterium, yet there are no the report that utilizes starch wastewater to prepare biological flocculant.
Summary of the invention
The purpose of this invention is to provide a kind of thermophilic ceramide bacillus BF-QSH7 and prepare high reactivity, the method for novel glycoprotein analog biological flocculant SPB-1 cheaply with it from starch wastewater, it can remedy the above-mentioned deficiency of prior art.
A kind of thermophilic ceramide bacillus is numbered BF-QSH7, it is characterized in that depositary institution is Wuhan China representative microbial culture collection center, and preserving number is for M.204033.
The application of above-mentioned thermophilic ceramide bacillus BF-QSH7 in preparation glycoprotein analog biological flocculant.
The above-mentioned thermophilic ceramide bacillus BF-QSH7 of a kind of usefulness prepares the method for glycoprotein analog biological flocculant, it is characterized in that adding in every liter of starch wastewater (NH
4)
2SO
40.5-4.0g, K
2HPO
41.0-3.0g and MgSO
40.1-0.2g as fermention medium, after the high-temperature steam sterilization, insert the seed culture medium that contains thermophilic ceramide bacillus and carry out fermentation culture, the fermentation culture temperature is 25-35 ℃, air flow is 0.51/min, stirring velocity is 300rpm, cultivate behind the 20-48h the fermented liquid centrifugation, collect thalline, with dilute hydrochloric acid extracting thalline, behind the extracting solution concentrating under reduced pressure, add the acetone or alcohol sedimentation, centrifugal collecting precipitation, vacuum obtains the pure product of biological flocculant in the dry crude product that promptly gets biological flocculant after ultrafiltration, dialysis, vacuum-drying.
The invention has the advantages that: the thermophilic ceramide bacillus BF-QSH7 of (1) institute seed selection can be the biological flocculant SPB-1 that the main raw material preparation has high flocculation activity with the starch wastewater, and fermentation time is short, and extraction process is simple, can reduce production costs greatly; (2) prepared biological flocculant SPB-1 is a kind of polymer glycoprotein analog material with a plurality of avtive spots, the flocculation activity height, and the flocculation object is extensive, and working conditions is extensive; (3) starch wastewater is behind the fermented extracted biological flocculant, and organic loading reduces greatly, helps subsequent disposal, for the processing of high concentration starch water provides convenience.
Description of drawings
Accompanying drawing 1 is the GC-MS figure of biological flocculant SPB-1 behind acidolysis and silanization.1,2,3,4 represent four kinds of contained in sample sugar components, i.e. D-glucose, D-semi-lactosi, D-seminose and talose respectively among the figure.
Accompanying drawing 2 is the MS collection of illustrative plates of D-Glucopyranose in MS figure and the standard spectrum storehouse of sugar component 1.A is the MS figure of sample among the figure, and b is corresponding standard MS figure, and matching rate is 92%.
Accompanying drawing 3 is the MS collection of illustrative plates of D-Galactopyranose in MS figure and the standard spectrum storehouse of sugar component 2.A is the MS figure of sample among the figure, and b is corresponding standard MS figure, and matching rate is 86%.
Accompanying drawing 4 is the MS collection of illustrative plates of D-Mannopyranose in MS figure and the standard spectrum storehouse of sugar component 3.A is the MS figure of sample among the figure, and b is corresponding standard MS figure, and matching rate is 91%.
Accompanying drawing 5 is the MS collection of illustrative plates of Talose in MS figure and the standard spectrum storehouse of sugar component 4.A is the MS figure of sample among the figure, and b is corresponding standard MS figure, and matching rate is 91%.
Accompanying drawing 6 is the infrared spectrogram of biological flocculant SPB-1
Embodiment
Thermophilic ceramide bacillus (S.thalpophilum) used among the present invention is numbered BF-QSH7, and it separates in the active sludge of sewage work, and nutrient agar is adopted in culture presevation.When this bacterial strain was cultivated on nutrient agar plate, bacterium colony was rounded, and neat in edge raises up, and smooth surface is wetting, placed under the room temperature to be yellow after 3 days; Thalline is a direct rod shape, no gemma, and atrichia has pod membrane; Thalline inner lipid sphingosine phosphatide.This bacterial strain major physiological biochemical characteristic sees Table 1.
Table 1 bacterial strain BF-QSH7 major physiological biochemical characteristic
| Biochemical character | The result | Biochemical character | The result |
| Urase | + | Glucose | + |
| Catalase | + | The D-pectinose | + |
| Oxydase | + | Fructose | + |
| O/F | -/+ | Semi-lactosi | + |
| The starch hydrolysis | + | Lactose | + |
| NO3 -1 reduction | - | Sucrose | + |
| The Vitamin C2 hydrolysis | + | Cellobiose | + |
| Lysine decarboxylase | - | Maltose | + |
| Ornithine decarboxylase | - | Trehalose | + |
| Arginine decarboxylase | - | Wood sugar | + |
| Indoles produces | - | H 2S produces | - |
| Acetate | - | Produce polymyxin B | + |
| Malonate | - | V-P | - |
During preparation glycoprotein analog biological flocculant, in every liter of tap water, add glucose 20.0g, yeast extract paste 0.5g, (NH
4)
2SO
42.0g, K
2HPO
45.0g, MgSO
40.2g, NaCl0.1g obtains seed culture medium, adds (NH in every liter of starch wastewater (take from Zhucheng, Shandong Province W-Gum factory of emerging trade company limited, the waste water quality analytical results sees Table 2)
4)
2SO
42.0g, K
2HPO
41.5g, MgSO
40.2g obtain fermention medium.Bacterial classification is transferred to above-mentioned seed culture medium from the nutrient agar medium inclined-plane, 200rpm, shaking table is cultivated 12h under 30 ℃ of conditions, makes somatic cells density reach 10
6-10
7Individual/ml, receive above-mentioned fermention medium with the inoculum size of 5% (v/v) then.Temperature is that 30 ℃, air flow are that 0.5l/min, stirring velocity are that 300rpm, pH are that 6.5-7.0, incubation time are 24h during fermentation culture.
The consumption of three kinds of used inorganic salt can be followed successively by 0.5-4.0g, 1.0-3.0g, 0.1-0.2g, wherein (NH in the fermention medium described in the present invention
4)
2SO
4Can use NH instead
4NO
3Or urea; Described inoculum size can be 2-10%.
Cultured fermented liquid is collected thalline with the centrifugal 20min of 10000rpm, every liter of fermented liquid can obtain the 15.8g wet thallus, with the hydrochloric acid extracting of 0.2mol/l three times, united extraction liquid, underpressure distillation to original volume 1/10th after, 4 ℃ add the cold acetone or the ethanol of 2 times of volumes down and place 4h, the centrifugal 10min of 6000rpm, collecting precipitation, vacuum-drying obtains yellow powder powder material, be flocculation agent crude product of the present invention, yield is every liter of fermented liquid 2.69g.
Gained flocculation agent crude product is suspended in the phosphate buffered saline buffer (pH is 7.4) of 100mM, ultrafiltration (10KDa) concentrates, and behind the distill water dialysis, lyophilize gets yellow powder powder material.The gained powdery substance is dissolved in the above-mentioned phosphate buffered saline buffer again, the same operation, ultrafiltration and concentration and lyophilize can obtain the pure product (SPB-1) of biological flocculant, and yield is every liter of fermented liquid 1.07g.
The total sugar content of biological flocculant SPB-1 of the present invention adopts sulfuric acid-phynol method to be determined as 81.9%, and protein content adopts the Coomassie brilliant blue method to be determined as 8.4%.With utilizing GC-MS to measure the composition and the content of monose behind SPB-1 acidolysis and the silanization, the results are shown in accompanying drawing 1,2,3,4,5.As can be seen from the figure, the component of sugar is D-glucose, D-semi-lactosi, D-seminose and talose, and their mass ratio is about 4: 5: 5: 1; Will be behind the SPB-1 alkaline hydrolysis measure wherein proteinic amino acid with automatic analyzer for amino acids and form, the result shows and contains aspartic acid (2.05%), Serine (2.45%), glycine (15.57%), L-Ala (16.49), Xie Ansuan (11.66), Isoleucine (3.17%), leucine (9.80%), tyrosine (1.29%), phenylalanine (5.44%), Methionin (9.74), Histidine (0.98%), arginine (6.01%), proline(Pro) multiple amino acids such as (15.3%) among the SPB-1; Results of IR is seen accompanying drawing 6.As can be seen from the figure, this biological flocculant is a kind of glycoprotein analog compound with a plurality of activated adsorption sites such as hydroxyl, amide group.The gel permeation chromatography result shows that its molecular weight is 3 * 10
6-6 * 10
6Between.
The resulting biological flocculant SPB-1 of the present invention kaolin suspension that can effectively flocculate, diatomite suspension, slime water, active sludge, when working concentration is 5.0mg/l, to the clearance of suspended particulate wherein all more than 95.0%; Various positively charged ions (comprise Na when coexisting
+, K
+, Mg
2+, Fe
2+, Ca
2+, Fe
3+, Al
3+) can not strengthen its flocculation activity, even restraining effect is arranged; This biological flocculant can keep its flocculation activity in the pH of broad scope, be 1-11 to the flocculation activity of kaolin suspension all more than 90% at pH.
The variation of waste water quality sees Table 2 before and after the fermentative preparation flocculation agent.As can be seen from Table 2, after steps such as fermentation culture, centrifugation thalline, extraction flocculation agent, COD
Cr, BOD
5Reach 85.0%, 86.0% and 97.7% respectively with the clearance of starch, organic loading significantly reduces in the water body, helps subsequent disposal.
Waste water quality before and after the table 2 fermentative preparation flocculation agent
| Project | COD Cr (mg/l) | BOD 5 (mg/l) | Starch (mg/l) | Total nitrogen (mg/l) | Total phosphorus (mg/l) |
| Before the processing | 15369 | 8967 | 7654 | 304 | 491 |
| After the processing | 2311 | 1254 | 179 | 102 | 96 |
| Clearance (%) | 85.0 | 86.0 | 97.7 | 66.4 | 80.4 |
Claims (3)
1. a thermophilic ceramide bacillus is numbered BF-QSH7, it is characterized in that depositary institution is Wuhan China representative microbial culture collection center, and preserving number is for M.204033.
2. the application of the described thermophilic ceramide bacillus BF-QSH7 of claim 1 in preparation glycoprotein analog biological flocculant.
3. a method for preparing the glycoprotein analog biological flocculant with the described thermophilic ceramide bacillus BF-QSH7 of claim 1 is characterized in that adding (NH in every liter of starch wastewater
4)
2SO
40.5-4.0g, K
2HPO
41.0-3.0g and MgSO
40.1-0.2g as fermention medium, after the high-temperature steam sterilization, insert the seed culture medium that contains thermophilic ceramide bacillus and carry out fermentation culture, the fermentation culture temperature is 25-35 ℃, air flow is 0.5l/min, stirring velocity is 300rpm, cultivate behind the 20-48h the fermented liquid centrifugation, collect thalline, with dilute hydrochloric acid extracting thalline, behind the extracting solution concentrating under reduced pressure, add the acetone or alcohol sedimentation, centrifugal collecting precipitation, vacuum-drying promptly gets the crude product of biological flocculant, obtains the pure product of biological flocculant after ultrafiltration, dialysis, vacuum-drying.
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|---|---|---|---|
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|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942407B (en) * | 2010-08-18 | 2012-01-25 | 无锡天易生物科技有限公司 | Producing strain and production method for producing microbial flocculant from wheat starch waste water |
| CN102260729B (en) * | 2011-06-24 | 2013-03-13 | 哈尔滨工业大学 | Bioflocculant fermentation method with mycelium pellet as vector |
| CN103146797B (en) * | 2013-02-26 | 2015-04-15 | 中国科学院重庆绿色智能技术研究院 | Compound biological flocculant and preparation method thereof |
| CN104445832B (en) * | 2014-12-18 | 2016-08-10 | 陕西科技大学 | Potato starch processing factory method of wastewater treatment |
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