CN102399823A

CN102399823A - Method for extracting bioflocculant

Info

Publication number: CN102399823A
Application number: CN2010102774691A
Authority: CN
Inventors: 丁国才
Original assignee: SHANGHAI HAIDI GARDENING CO Ltd
Current assignee: SHANGHAI HAIDI GARDENING CO Ltd
Priority date: 2010-09-09
Filing date: 2010-09-09
Publication date: 2012-04-04

Abstract

The invention relates to a method for extracting a bioflocculant. The method is characterized by extracting the flocculant from activated sludge, has the advantages of wide source and low cost, comprising the following steps: screening the activated sludge and extracting bacterial strains with best activity, culturing in a glucose medium, and then carrying out extraction on the supernatant liquor in the medium by acetone process, ethanol process or CTAB process. The obtained flocculant has high activity, high yield, no secondary pollution to the environment, and very good potential of large scale production.

Description

A kind of method of extracting biological flocculant

Technical field

The present invention relates to a kind of process for extracting from biological flocculant.

Background technology

Microbial flocculant be mikrobe under Incubation Condition, born of the same parents' extra-metabolite that growth metabolism to certain phase produces with flocculation activity, staple is gp, polysaccharide, protein, DNA etc.The later stage eighties 20th century; The grand youth of the storehouse root of Japan filters out rhodococcus erythropolis S-1 (R Erythropolis S-1) bacterial strain; Will be by the microbial flocculant called after NOC-1 of this bacterial strain generation; Be the best microbial flocculant of finding so far of flocculating effect, various wastewater is all had good flocculation turbidity removal effect.Microbial flocculant is different with traditional chemical floc, not only has good flocculation sediment performance, and safety, and is nontoxic, biodegradable, and ecotope is not had a negative impact yet; Simultaneously, the microbe species that can produce flocculation agent is many, and growth is fast, is easy to take the biotechnology means to realize industrialization, thereby has vast potential for future development.The biggest obstacle that microbial flocculant is used is that consumption is big, cost is high, so screening high-effective microorganism bacterium for producing flocculant improves flocculation activity, reduces flocculation agent consumption and cost, is the problem that present biological flocculant need solve.

Summary of the invention

The present invention provides a kind of flocculation activity high, and material is convenient, a kind of method of extracting biological flocculant with low cost, and its technical scheme is following:

A kind of method of extracting biological flocculant may further comprise the steps: screen from the bacterial strain of active sludge (1), filters out the best bacterial strain of flocculation activity;

(2) bacterial strain in the step (1) is cultivated in dextrose culture-medium cultivated in 22～88 hours;

(3) adopt acetone method, Ethanol Method or CTAB method to handle the nutrient solution in the step (2), obtain the product of flocculation agent.

The method of aforesaid extraction biological flocculant is characterized in that, the condition of from active sludge, extracting in the step (1) is: after the active sludge dilution; Put 30 ℃ of constant temperature shaking tables, 160r/min enrichment culture 3d adopts the dilution plate coating method to obtain single bacterium colony; Number behind the purifying; Respectively bacterial strain behind the purifying is connect in the people 100mL dextrose culture-medium again, 30 ℃, 160r/min shake-flask culture 3d; The gained nutrient solution carries out the rough determination of flocculation activity, and flocculation activity the higher person is the bacterial strain of produce flocculant.

The method of aforesaid extraction biological flocculant is characterized in that, the consisting of of the dextrose culture-medium in step (1) and (2): glucose 20g, KH ₂PO ₄0.2g, K ₂HPO ₄0.5g, (NH ₄) ₂SO ₄0.2g, NaCl 0.1g, urea 0.5g, yeast extract paste 0.5g, MgSO47H ₂O 0.2g, 2% agar, 1000mL water, transferring pH is 6.0～9.0.

The method of aforesaid extraction biological flocculant, wherein the incubation time in the step (2) is 66 hours.

The method of aforesaid extraction biological flocculant, wherein said flocculation activity measuring method is following: take by weighing 0.1g kaolin, add zero(ppm) water to 200mL; Process the kaolin suspension, get 1.5～3mL liquid to be measured and join in the 200mL kaolin suspension, stir 10min at a slow speed; Leave standstill 5min, survey supernatant absorbancy B at the 550nm place, replace liquid to be measured as contrast with deionized water; Survey absorbancy with method, flocculating rate=(A-B)/A100%.

The method of aforesaid extraction biological flocculant, the PH of wherein said dextrose culture-medium is 7.5.

The method of aforesaid extraction biological flocculant, wherein the nutrient solution in the step (3) adopts the CTAB method to extract.

Adopt the present invention to extract biological flocculant, easy and simple to handle, to draw materials conveniently, the flocculation agent flocculation activity of extraction is unusual height also, and cost is low, is suitable for extensive use, also can not produce environment after it uses and pollute, and the protection environment is also played a role.

Description of drawings

Fig. 1 is effective flocculation composition distribution plan of the flocculation activity that from active sludge, extracts.

Fig. 2 is the flocculation activity contrast of the flocculation composition cultivated in the different dextrose culture-medium of original PH.

Fig. 3 is the flocculation activity contrast of different culture time to the flocculation composition influence.

Fig. 4 is that the flocculant dosage volume(tric)fraction is not simultaneously to the influence of flocculation activity.

Fig. 5 is the productive rate contrast of the flocculation agent that obtains of three kinds of process for extracting.

Embodiment

To combine specific embodiment of the present invention that technical scheme is carried out detailed explanation below:

The preparation embodiment of flocculation agent:

Bacterial classification derives from Sewage Plant aeration tank, Yangpu District Sector East, Shanghai active sludge, basic medium: beef-protein medium; Screening culture medium (being also referred to as dextrose culture-medium): with glucose 20g, KH ₂PO ₄0.2g, K ₂HPO ₄0.5g, (NH ₄) ₂SO ₄0.2g, NaCl 0.1g, urea 0.5g, yeast extract paste 0.5g, MgSO47H ₂O0.2g, (2% agar) joins in the 1000mL water, allocates pH respectively and be 6.0～9.0 several parts of substratum, 112 ℃ of sterilization 30min.

One. bacterial screening: after the active sludge dilution, put 30 ℃ of constant temperature shaking tables, 160r/min enrichment culture 3d adopts the dilution plate coating method to obtain single bacterium colony, numbers behind the purifying.Respectively bacterial strain behind the purifying is connect in the people 100mL screening culture medium again, 30 ℃, 160r/min shake-flask culture 3d, the gained nutrient solution carries out the rough determination of flocculation activity, and flocculation activity the higher person is the produce flocculant bacterial strain.Flocculation activity is measured: take by weighing 0.1g kaolin, add zero(ppm) water to 200mL, process the kaolin suspension; Get 2mL liquid to be measured (clear liquid that promptly from nutrient solution, extracts) and join in the 200mL kaolin suspension, stir 10min at a slow speed, leave standstill 5min, survey supernatant absorbancy B at the 550nm place, replace liquid to be measured as contrast, survey absorbance A with method with deionized water.Flocculating rate=(A-B)/A100%.Through the screening to active sludge, present embodiment screens the higher bacterial strain of 1 strain flocculation activity, and the flocculation activity of its culture reaches 44.7%.

Two. the test that effective ingredient distributes:, compare the flocculating rate of nutrient solution, centrifugal back supernatant, centrifugal back bacteria suspension respectively with the centrifugal 20min of nutrient solution 3000r/min in the step 1.Its result is as shown in Figure 1, such as figure effective flocculation composition of demonstration culture mainly concentrate in the supernatant, bacteria suspension does not almost have flocculation activity, therefore, in following step, the mensuration of flocculation activity is all got the supernatant of culture.

Three. culture condition optimization:, respectively initial pH of substratum and incubation time are measured the influence of flocculating rate with carrying out shake-flask culture after the bacterial classification seed culture again.

Divide and prepare pH 6.0 in addition, pH 6.5, and pH 7.0, and pH 7.5; PH8.0, the dextrose culture-medium of pH8.5 and pH9.0, shake-flask culture 3d surveys the flocculating rate result and sees Fig. 2; Visible by Fig. 2, this bacterial strain is adapted at growth and synthetic agglutinating matter under the pH partial neutral condition, and when pH 7.5, flocculating rate is maximum.Under acidic conditions, during like pH 6.0, flocculating rate is merely 32.2%, equally also is unfavorable for the generation of flocculation agent under the alkaline condition, and as when the pH9.0, flocculating rate is merely 36.0%.Drawn by Fig. 2, the pH of this bacterial strain suitable growth is 7.0～7.5, and flocculating rate is maximum during pH7.5, and therefore, in subsequent experimental, the initial pH of substratum is decided to be 7.5.

Bacterial classification after the screening is carried out shake-flask culture, be respectively 22h at incubation time, 44h, 66h during 88h, surveys its flocculating rate result and sees Fig. 3.Visible by Fig. 3, be 0～22h at incubation time, microorganism growth is rapid, and metabolism is vigorous, and the culture flocculation activity increases with incubation time and improves; 22～66h flocculating rate slightly raises, but changes not quite, and flocculating rate is the highest when cultivating 66h; Owing to the metabolic consumption of microorganism growth, the flocculating rate of nutrient solution begins to reduce behind the 66h.Therefore, the Best Times of this experiment cultivation is set at 66h.

Measure the flocculant dosage volume(tric)fraction respectively and be 0.75%, 1%, 1.25% and 1.5% o'clock flocculating rate, the result sees Fig. 4.Can be known by Fig. 4: the supernatant dosage is best at 1.00%～1.25% o'clock flocculating effect, reach 59.5%. less than or during greater than this scope, flocculating effect all decreases.

Four. extract with following three kinds of process for extracting respectively:

(1) the acetone method medium centrifugal separates (5000r/min, centrifugal 25min) removal deposition (thalline and foreign material); Add the acetone of 1.5 times of volumes in the supernatant, 4 ℃ of placements are spent the night; 12000r/min, centrifugal 10min removes supernatant; The ether washing precipitation; Vacuum-drying obtains the flocculation agent raw product.

(2) the Ethanol Method medium centrifugal separates (5000r/min, centrifugal 25min) removal deposition (thalline and foreign material); The absolute ethyl alcohol that adds 1.5 times of volume precoolings in the supernatant, mixing, centrifugal, collecting precipitation; 70% washing with alcohol deposition; Vacuum-drying obtains the flocculation agent raw product.

(3) cetyl trimethylammonium bromide (being CTAB) method medium centrifugal separates (5000r/min, centrifugal 25min) removal deposition (thalline and foreign material); Slow adding concentration is 10% CTAB in the above-mentioned stillness of night, does not separate out up to there being deposition; Centrifugal, collecting precipitation; Deposition is dissolved in the NaCl solution of 2mol/L, the absolute ethyl alcohol that adds 2～4 times of volumes precipitates (not separating out up to there being deposition); Wash with 75% ethanol, absolute ethyl alcohol, ether respectively; Vacuum-drying obtains the flocculation agent raw product.

The contrast in the thick flocculation activity of producing of flocculation agent to three kinds of process for extracting obtain is as shown in Figure 4; Dosage is under the situation of 3mg/L, and respectively to the acetone extraction method, the flocculation agent bullion that ethanol extraction method and CTAB extraction method obtain carries out the mensuration of flocculating rate; Flocculating rate result is followed successively by 66.0%; 67.4% and 71.8%, the raw product flocculation activity that obtains like visible 3 kinds of methods among the figure is more or less the same, and it is higher relatively that the CTAB method obtains the flocculation activity of flocculation agent.

The bullion productive rate of the flocculation agent that three kinds of process for extracting extract as shown in Figure 5, the acetone extraction method, in ethanol extraction method and the CTAB3 kind extraction method, the productive rate of the flocculation agent that the CTAB extraction method obtains is the highest, reaches 1.33g/L.

The flocculation agent productive rate height that obtains that from microbial culture medium, extracts of the present invention has higher flocculation activity, and can not cause secondary pollution to environment, has boundless application prospect.

Claims

1. method of extracting biological flocculant, may further comprise the steps: the bacterial strain that extract from active sludge (1) screens, and in dextrose culture-medium, after cultivating, filters out the best bacterial strain of flocculation activity; (2) bacterial strain in the step (1) is cultivated in dextrose culture-medium cultivated in 22～88 hours; (3) adopt acetone method, Ethanol Method or CTAB method to handle the nutrient solution in the step (2), obtain flocculant product.

2. like the method for the extraction biological flocculant in the claim 1, it is characterized in that the condition of from active sludge, extracting in the step (1) is: after the active sludge dilution; Put 30 ℃ of constant temperature shaking tables, 160r/min enrichment culture 3d adopts the dilution plate coating method to obtain single bacterium colony; Number behind the purifying; Respectively the bacterial strain behind the purifying is connect in the people 100mL dextrose culture-medium again, 30 ℃, 160r/min shake-flask culture 3d; The gained nutrient solution carries out the rough determination of flocculation activity, and flocculation activity the higher person is the bacterial strain of produce flocculant.

3. like the method for the extraction biological flocculant in claim 1 or 2, it is characterized in that the consisting of of the dextrose culture-medium in step (1) and (2): glucose 20g, KH ₂PO ₄0.2g, K ₂HPO ₄0.5g, (NH ₄) ₂SO ₄0.2g, NaCl 0.1g, urea 0.5g, yeast extract paste 0.5g, MgSO47H ₂O 0.2g, 2% agar, 1000mL water, transferring pH is 6.0～9.0.

4. like the method for the extraction biological flocculant in the claim 1, it is characterized in that: the incubation time in the step (2) is 66 hours.

5. like the method for the extraction biological flocculant in the claim 2, it is characterized in that described flocculation activity measuring method is following: take by weighing 0.1g kaolin; Add zero(ppm) water to 200mL, process the kaolin suspension, get 1.5～3mL liquid to be measured and join in the 200mL kaolin suspension; Stir 10min at a slow speed, leave standstill 5min, survey supernatant absorbancy B at the 550nm place; Replace liquid to be measured as contrast with deionized water, survey absorbancy with method, flocculating rate=(A-B)/A100%.

6. the method for extraction biological flocculant as claimed in claim 3 is characterized in that: the PH of described dextrose culture-medium is 7.5.

7. the method for extraction biological flocculant as claimed in claim 1 is characterized in that: the nutrient solution in the step (3) adopts the cetyl trimethylammonium bromide method to extract.