CN102181483A

CN102181483A - Preparation method of microbial flocculant

Info

Publication number: CN102181483A
Application number: CN201110067709XA
Authority: CN
Inventors: 肖弘
Original assignee: 肖弘
Priority date: 2011-03-21
Filing date: 2011-03-21
Publication date: 2011-09-14

Abstract

The invention discloses a preparation method of a microbial flocculant. The preparation method comprises the following steps: screening flocculant-producing strains from activated sludge, inoculating the flocculant-producing strains in seed culture fluid to perform seed culture, then inoculating in fermentation culture fluid to perform fermentation culture at 25-35 DEG C; then centrifugalizing the fermentation culture fluid to obtain concentrated solution through rotary evaporation, adding acetone to obtain precipitate; and finally centrifugalizing the precipitate, and performing vacuum drying to obtain the microbial flocculant. In the preparation method of the microbial flocculant, activated sludge is used as bacteria source and screening is adopted to obtain the flocculant-producing strains; and the yield is high and the flocculation activity is higher.

Description

The preparation method of microbial flocculant

Technical field

The present invention relates to a kind of preparation method of microbial flocculant.

Background technology

Microbial flocculant is the meta-bolites that a class is produced by microorganism or its secretory product, it is to utilize microbial technique, by microbial fermentations such as bacterium, fungi, extraction, refining getting, be water conditioner with efficient, nontoxic, non-secondary pollution of Biodegradable and security.Because microbial flocculant can overcome inorganic polymer and the inherent defective of synthetic organic polymer flocculation agent own, finally realize non-pollution discharge, so the research of microbial flocculant is just becoming the important topic of world today's flocculation agent aspect research.How screening the high-effective microorganism bacterium for producing flocculant, improve flocculation activity, reduce flocculation agent consumption and cost, is the key that microbial flocculant is applied.

Summary of the invention

The object of the present invention is to provide a kind of preparation method of microbial flocculant.

The preparation method of microbial flocculant of the present invention comprises:

(a), screening produce flocculant bacterial strain from active sludge;

(b), described produce flocculant inoculation is carried out seed culture in seed culture fluid, be inoculated into fermentation culture in the fermentation culture then, leavening temperature is 25 ~ 35 ℃;

(c), with the fermentation culture centrifugation, after rotary evaporation obtains concentrated solution, add acetone and obtain throw out;

(d), described throw out is obtained described microbial flocculant through centrifugation and vacuum-drying.

According to the present invention, described seed culture medium contains 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v %, six ferrous sulfate hydrate ammoniums, 0.01～0.2 w/v % sodium-chlor, all the other are water, pH is 5.5 ~ 8.5.

According to the present invention, described fermention medium contains 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v % bitter salt, 0.01～0.2 w/v % Repone K, all the other are water, pH is 5.5 ~ 8.5.

According to the present invention, described fermentation is carried out in fermentor tank, and mixing speed is 200～300rpm, and air flow is 0.5～1.5L/min, and fermentation time is 15 ~ 20hr.

According to the present invention, the centrifugal condition of described steps d is at 6000 ~ 8000r/min, centrifugal 20 ~ 30min; Described vacuum drying condition is that vacuum tightness is 0.04 ~ 0.06Mpa, and vacuum drying temperature is 58 ~ 62 ℃.

According to the present invention, described preparation method comprises that also the microbial flocculant that steps d is obtained carries out the purified step: described microbial flocculant is dissolved in forms lysate in the sterilized water, the mass ratio of described flocculation agent and described water is 1:80 ~ 120, add chloroform, the volume ratio of described lysate and described chloroform is 1:1 ~ 2, concussion back standing demix is got supernatant, pack in the dialysis tubing, with deionized water 35 ~ 45 ℃ the dialysis 30 ~ 50hr, liquid in the dialysis tubing is added 3 ~ 5 times of volume of ethanol, leave standstill the generation flocculate precipitate, in the centrifugal 15 ~ 25min of 4000 ~ 5000r/min, abandoning supernatant, is 0.04 ~ 0.06Mpa in vacuum tightness, and vacuum drying temperature is vacuum-drying 4 ~ 8hr under 58 ~ 62 ℃ the condition.

The preparation method of microbial flocculant of the present invention is the bacterium source with the active sludge, and by the bacterial strain of screening acquisition produce flocculant, the productive rate height has higher flocculation activity.

Embodiment

Further specify the present invention below in conjunction with embodiment, but the scope of the inventive method is not limited to following example.

The prescription of the substratum that adopts in following examples is as follows:

Screening culture medium: 20g glucose, 0.2g KH ₂P0 ₄, 0.5g K ₂HP0 ₄, 0.2 g (NH ₄) ₂S0 ₄, 0.1 g NaCI, 0.5 g urea, 0.5 g yeast extract paste, 0.2 gMgS0 ₄7H ₂0, (2% agar) joins in the 1 000 mL water, and transferring pH is 7.0,112 ℃ of sterilization 30 min.

Seed culture medium: 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v %, six ferrous sulfate hydrate ammoniums, 0.01～0.2 w/v % sodium-chlor, all the other are water, pH is 5.5 ~ 8.5.

Fermention medium: 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v % bitter salt, 0.01～0.2 w/v % Repone K, all the other are water, pH is 5.5 ~ 8.5.

The screening of embodiment 1 produce flocculant bacterial strain

Get the active sludge in the Sewage Plant aeration tank, put 28 ~ 32 ℃ of constant temperature shaking tables, 140 ~ 160r/min enrichment culture 2 ~ 4 days adopts the dilution plate coating method to obtain single bacterium colony, numbering.Respectively bacterial strain behind the purifying is inserted in the 100 mL screening culture medium again, put 28 ~ 32 ℃, 140 ~ 160r/min shake-flask culture 2 ~ 4 days, adopt following method the gained nutrient solution to be carried out the rough determination of flocculation activity.The result is as shown in table 1.

Take by weighing 0.1g kaolin, add distilled water to 200 mL, make the kaolin suspension; Get 2 mL liquid to be measured and join in the 200 mL kaolin suspensions, stir 10 min at a slow speed, leave standstill 5mm, survey supernatant liquor absorbancy B at 550 nm places, replace liquid to be measured in contrast, survey absorbance A with method with deionized water.

Table 1 The selection result

As shown in Table 1, by the screening to active sludge, the flocculation activity of No. 6 bacterial strains reaches 42.5%, for the produce flocculant bacterial strain, is used for follow-up test.

The preparation of embodiment 2 flocculation agents

The produce flocculant inoculation that embodiment 1 screening is obtained places shaking table to carry out seed culture in the Erlenmeyer flask that seed culture fluid is housed, and wherein, shaking speed is 200rpm, and temperature is 28 ℃, and incubation time is 46h.Be inoculated into fermentation culture in the fermentation culture then, leavening temperature is 25 ℃, and mixing speed is 200rpm, and air flow is 0.5L/min, and fermentation time is 15hr.Fermentation culture is separated at 6000r/min, centrifugal 20 ~ 30min, adds acetone obtain throw out after rotary evaporation obtains concentrated solution, and the volume ratio of concentrated solution and acetone is 1:1; The throw out warp that obtains being separated at 6000r/min, centrifugal 20min, abandon supernatant, is 0.04Mpa in vacuum tightness, and temperature is that vacuum-drying obtains microbial flocculant under 58 ℃ of conditions, and productive rate is 1.12g/L, and flocculation activity is 43.7%.

The preparation of embodiment 3 flocculation agents

The produce flocculant inoculation that embodiment 1 screening is obtained places shaking table to carry out seed culture in the Erlenmeyer flask that seed culture fluid is housed, and wherein, shaking speed is 230rpm, and temperature is 30 ℃, and incubation time is 50h.Be inoculated into fermentation culture in the fermentation culture then, leavening temperature is 35 ℃, and mixing speed is 300rpm, and air flow is 1.5L/min, and fermentation time is 20hr.Fermentation culture 8000r/min, centrifugal 30min are separated, add acetone obtain throw out after rotary evaporation obtains concentrated solution, and the volume ratio of concentrated solution and acetone is 1:2; The throw out warp that obtains being separated at 8000r/min, centrifugal 30min, abandon supernatant, is 0.06Mpa in vacuum tightness, and temperature is that vacuum-drying obtains microbial flocculant under 62 ℃ of conditions, and productive rate is 1.43g/L, and flocculation activity is 44.8%.

Making with extra care of embodiment 4 flocculation agents

The microbial flocculant that embodiment 2 is obtained is dissolved in and forms lysate in the sterilized water, the mass ratio of described flocculation agent and described water is 1:80 ~ 120, add chloroform, the volume ratio of described lysate and described chloroform is 1:1 ~ 2, concussion back standing demix is got supernatant, pack in the dialysis tubing, at 35 ~ 45 ℃ of dialysis 30 ~ 50hr, the liquid in the dialysis tubing is added 3 ~ 5 times of volume of ethanol with deionized water, leave standstill the generation flocculate precipitate, in the centrifugal 15 ~ 25min of 4000 ~ 5000r/min, abandoning supernatant, is 0.04 ~ 0.06Mpa in vacuum tightness, and vacuum drying temperature is vacuum-drying 4 ~ 8hr under 58 ~ 62 ℃ the condition, obtain the flocculation agent elaboration, flocculation activity is 46.2%.

Claims

1. the preparation method of a microbial flocculant is characterized in that, comprising:

(a), screening produce flocculant bacterial strain from active sludge;

2. preparation method as claimed in claim 1 is characterized in that, described seed culture medium contains 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v %, six ferrous sulfate hydrate ammoniums, 0.01～0.2 w/v % sodium-chlor, all the other are water, pH is 5.5 ~ 8.5.

3. preparation method as claimed in claim 1 is characterized in that, described fermention medium contains 0.5～2.0 w/v % Zulkovsky starch, 0.1～1.0 w/v %NaNO ₃, 0.05～0.5 w/v %K ₂HPO ₄, 0.01～0.2w/v % bitter salt, 0.01～0.2 w/v % Repone K, all the other are water, pH is 5.5 ~ 8.5.

4. preparation method as claimed in claim 1 is characterized in that described fermentation is carried out in fermentor tank, mixing speed is 200～300rpm, and air flow is 0.5～1.5L/min, and fermentation time is 15 ~ 20hr.

5. preparation method as claimed in claim 1 is characterized in that, the centrifugal condition of described steps d is at 6000 ~ 8000r/min, centrifugal 20 ~ 30min; Described vacuum drying condition is that vacuum tightness is 0.04 ~ 0.06 Mpa, and vacuum drying temperature is 58 ~ 62 ℃.

6. preparation method as claimed in claim 1, it is characterized in that, described preparation method comprises that also the microbial flocculant that steps d is obtained carries out the purified step: described microbial flocculant is dissolved in forms lysate in the sterilized water, the mass ratio of described flocculation agent and described water is 1:80 ~ 120, add chloroform, the volume ratio of described lysate and described chloroform is 1:1 ~ 2, concussion back standing demix is got supernatant, pack in the dialysis tubing, with deionized water 35 ~ 45 ℃ the dialysis 30 ~ 50hr, liquid in the dialysis tubing is added 3 ~ 5 times of volume of ethanol, leave standstill the generation flocculate precipitate,, abandon supernatant in the centrifugal 15 ~ 25min of 4000 ~ 5000r/min, in vacuum tightness is 0.04 ~ 0.06Mpa, and vacuum drying temperature is vacuum-drying 4 ~ 8hr under 58 ~ 62 ℃ the condition.