阅读说明：本技术 一种从活性污泥中提取海藻多糖的方法 (Method for extracting algal polysaccharide from activated sludge ) 是由 薛蔚琦 孙临泉 田泽民 姜维 肖倩 屈茂会 于 2020-12-16 设计创作，主要内容包括：本发明公开了一种从活性污泥中提取海藻多糖的方法,涉及活性污泥回收技术领域。本发明提供的从活性污泥中提取海藻多糖的方法,以污水处理厂的污水生物处理过程所依赖并产生的活性污泥为原料,对活性污泥微生物细胞进行高效破壁,使微生物细胞的内部物质被释放,再优化提取方法,得到纯化的海藻多糖(包括藻石酸、褐藻多糖、卡拉胶及肝素等)。(The invention discloses a method for extracting algal polysaccharide from activated sludge, and relates to the technical field of activated sludge recovery. The method for extracting algal polysaccharide from activated sludge provided by the invention takes the activated sludge depended and generated in the biological sewage treatment process of a sewage treatment plant as a raw material, carries out high-efficiency wall breaking on activated sludge microbial cells to release internal substances of the microbial cells, and optimizes the extraction method to obtain purified algal polysaccharide (comprising alginic acid, fucoidan, carrageenan, heparin and the like).)

1. A method for extracting algal polysaccharides from activated sludge is characterized by comprising the following steps:

s1, carrying out high-efficiency wall breaking on microbial cells in the activated sludge to obtain mud-water mixed liquor containing endogenous substances;

s2, performing centrifugal filtration on the mud-water mixed solution containing the endogenous substances, and removing solid-phase mud cakes to obtain a liquid-phase component;

s3, adding a precipitator into the liquid phase component until no precipitate is generated, and performing centrifugal filtration to obtain an alginate precipitate and a supernatant;

s4, adding ethanol into the supernatant of S3, centrifuging and filtering after the flocculent precipitate is complete, and keeping the flocculent precipitate;

s5, repeatedly washing the flocculent precipitate with a saturated NaCl solution and ethanol until the flocculent precipitate is dissolved in the saturated NaCl solution and no insoluble impurities exist, adding ethanol into the saturated NaCl solution in which the flocculent precipitate is dissolved until the precipitate is complete, and centrifugally filtering to obtain algal polysaccharide precipitate;

and S6, adding distilled water into the algal polysaccharide precipitate of S5 until the algal polysaccharide precipitate is completely dissolved, separating solution components by using steric exclusion chromatography, comparing the peak-off time of the algal polysaccharide, the carrageenan and the heparin sodium under the same operation condition, collecting liquid phase separation components, and drying to obtain the algal polysaccharide, the carrageenan and the heparin sodium solid respectively.

2. The method for extracting algal polysaccharides from activated sludge according to claim 1, wherein the specific operation of performing high-efficiency wall breaking on the microbial cells in the activated sludge in the step S1 comprises the following steps:

s11, concentrating the activated sludge to mixed liquid with suspended solid concentration of 3000-;

s12, centrifuging the mixed solution of S11, discarding the upper liquid phase, adding distilled water with the same volume into the solid-phase mud cake, shaking uniformly, centrifuging again, discarding the upper liquid phase, repeating the mud washing step for 2 to 4 times, and reserving the solid-phase mud cake after centrifuging;

s13, adding distilled water into the solid-phase mud cake of S12 according to a mud-water volume ratio of 1:5(v/v) and shaking uniformly to obtain a mixed solution, heating the mixed solution to 30-50 ℃, and electrifying to break the wall of the mixed solution for 10-30 minutes to obtain the mud-water mixed solution containing the endogenous substances.

3. The method for extracting algal polysaccharides from activated sludge according to claim 2, wherein the step of electrically breaking the walls is to apply a direct current of 5-30V to the mixed solution in a cycle of "10 seconds, 5 seconds of intermittence, and 10 seconds of re-energization".

4. The method for extracting algal polysaccharides from activated sludge according to claim 1, wherein a concentration step is further included between the step S2 and the step S3, and specifically, the liquid phase component obtained in the step S2 is subjected to ultrafiltration until the volume of the liquid phase is concentrated to 50-60% of the volume before ultrafiltration.

5. The method of extracting algal polysaccharides from activated sludge according to claim 1, wherein in the step S3, the precipitant is calcium chloride.

6. The method for extracting algal polysaccharides from activated sludge according to claim 1, wherein the mass fraction of the ethanol is 98%.

7. The method of extracting algal polysaccharide from activated sludge according to claim 1, wherein in the step S4, ethanol is added to the supernatant of S3 until the mass fraction of ethanol in the solution is 60%.

8. The method of claim 7, wherein the step S4 further comprises adding ethanol and standing the solution at 0 deg.C for 1-2 hours.

9. The method for extracting algal polysaccharide from activated sludge as set forth in claim 1, wherein the rotation speed of the centrifugal filtration is 4000-9000 rpm.

10. The method of claim 1, wherein the flocculent precipitate is repeatedly washed with saturated NaCl solution and ethanol in step S5, and the following steps are performed:

s51, adding a NaCl solution into the flocculent precipitate until the flocculent precipitate is completely dissolved, centrifuging, discarding the impurity precipitate, and keeping a liquid phase after centrifuging;

s52, adding ethanol into the liquid phase after centrifugation until the precipitation is complete;

repeating S51-S52 until the flocculent precipitate is dissolved in the saturated NaCl solution and no insoluble impurities exist, which indicates that the washing is finished.

Technical Field

The invention relates to the technical field of activated sludge recovery treatment, in particular to a method for extracting algal polysaccharide from activated sludge.

Background

Algal polysaccharides are intercellular polysaccharides with complex composition structure, various varieties and wide application, and are known to exist mainly in algae and a few animal tissues (lung lobes of cattle, gastric mucosa of pigs and the like) and exist in a few halophilic plants and microorganisms at the same time. The chemical properties and biological activity of algal polysaccharides are influenced by chemical composition, structure and sulfation degree, and the main chemical properties and biological activity are shown as follows: gel thickening property, oxidation resistance, anticoagulation property, angiogenesis inhibition (antitumor property), immunoregulation property, anti-HSV and anti-HIV virus property. Algal polysaccharides can be widely used as industrial additives, food and cosmetic additives, and pharmaceutical intermediates according to their high or low purity. At present, the widely used algal polysaccharides mainly include: alginate, fucoidan, carrageenan, heparin sodium, etc.

Because the chemical composition and the structure of the algal polysaccharide are complex, the algal polysaccharide can not be produced in a large scale in an artificial synthesis mode. Currently more than 90% of algal polysaccharide production worldwide relies on direct extraction from algae, with the remainder totally relying on animal tissue extraction. Although the artificial seaweed cultivation technology is rapidly developed since the end of the fifties of the twentieth century, a large amount of raw materials are provided for the production of seaweed polysaccharide, the seaweed cultivation has high dependence on weather and seawater conditions, the suitable cultivation area is limited, the seaweed cultivation cost is high, the seaweed collection completely depends on manpower, the extraction rate of the existing extraction method is limited, the global seaweed polysaccharide market is always in a state of supply and demand, and the price of seaweed polysaccharide products is very high. In addition, a large amount of cultivation waste water generated by artificial seaweed cultivation also becomes a considerable environmental problem in seaweed cultivation.

The activated sludge is a byproduct which is depended on by sewage biological treatment and is produced in large quantity, the essence of the activated sludge is a microbial aggregate which is propagated in large quantity by utilizing nutrient elements such as carbon, nitrogen, phosphorus and the like in the sewage, and the activated sludge is named as the activated sludge because the microbial community is complex, the propagation quantity is large, the appearance is tan and has a sludge shape. For a long time, activated sludge is regarded as a large amount of waste generated in the biological treatment process of sewage, and the treatment of activated sludge is also aimed at 'harmlessness and reduction', and the main treatment modes comprise: sanitary landfill, incineration, anaerobic digestion, aerobic composting and the like. But many recyclable materials in the activated sludge have been ignored.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for extracting algal polysaccharide from activated sludge, which solves the problem of recovery treatment of activated sludge on one hand and alleviates the problem of single production mode of algal polysaccharide at present on the other hand.

In order to solve the above problems, the present invention proposes the following technical solutions:

a method for extracting algal polysaccharides from activated sludge comprises the following steps:

s1, carrying out high-efficiency wall breaking on microbial cells in the activated sludge to obtain mud-water mixed liquor containing endogenous substances;

s2, performing centrifugal filtration on the mud-water mixed solution containing the endogenous substances, and removing solid-phase mud cakes to obtain a liquid-phase component;

s3, adding a precipitator into the liquid phase component until no precipitate is generated, and performing centrifugal filtration to obtain an alginate precipitate and a supernatant;

s4, adding ethanol into the supernatant of S3, centrifuging and filtering after the flocculent precipitate is complete, and keeping the flocculent precipitate;

s5, repeatedly washing the flocculent precipitate with a saturated NaCl solution and ethanol until the flocculent precipitate is dissolved in the saturated NaCl solution and no insoluble impurities exist, adding ethanol into the saturated NaCl solution in which the flocculent precipitate is dissolved until the precipitate is complete, and centrifugally filtering to obtain algal polysaccharide precipitate;

and S6, adding distilled water into the algal polysaccharide precipitate of S5 until the algal polysaccharide precipitate is completely dissolved, separating solution components by using steric exclusion chromatography, comparing the peak-off time of the algal polysaccharide, the carrageenan and the heparin sodium under the same operation condition, collecting liquid phase separation components, and drying to obtain the algal polysaccharide, the carrageenan and the heparin sodium solid respectively.

The further technical scheme is that the solid of the alginic acid is obtained by collecting and drying the precipitate of the alginic acid salt in the step S3.

The further technical scheme is that in the step S1, the specific operation of performing high-efficiency wall breaking on microbial cells in the activated sludge comprises the following steps:

s11, concentrating the activated sludge to mixed liquid with suspended solid concentration of 3000-;

s12, centrifuging the mixed solution of S11, discarding the upper liquid phase, adding distilled water with the same volume into the solid-phase mud cake, shaking uniformly, centrifuging again, discarding the upper liquid phase, repeating the mud washing step for 2 to 4 times, and reserving the solid-phase mud cake after centrifuging;

s13, adding distilled water into the solid-phase mud cake of S12 according to a mud-water volume ratio of 1:5(v/v) and shaking uniformly to obtain a mixed solution, heating the mixed solution to 30-50 ℃, and electrifying to break the wall of the mixed solution for 10-30 minutes to obtain the mud-water mixed solution containing the endogenous substances.

The further technical scheme is that the operation of electrifying and wall breaking is specifically that 5-30V direct current is introduced into the mixed solution according to the period of electrifying for 10 seconds, intermittence for 5 seconds and electrifying for 10 seconds again.

The method further comprises a concentration step between the step S2 and the step S3, specifically, the liquid phase component obtained in the step S2 is subjected to ultrafiltration until the volume of the liquid phase is concentrated to 50-60% of the volume before ultrafiltration.

The technical scheme is that in the step S3, the precipitator is calcium chloride.

The further technical scheme is that the mass fraction of the ethanol is 98%.

In the step S4, ethanol is added to the supernatant of S3 until the mass fraction of ethanol in the solution is 60%.

The further technical scheme is that the step S4 further comprises the step of standing the solution for 1-2 hours at 0 ℃ after adding ethanol.

The further technical proposal is that the rotational speed of the centrifugal filtration is 4000-9000 rpm.

The further technical scheme is that in the step S5, the flocculent precipitate is repeatedly washed with a saturated NaCl solution and ethanol, specifically the following operations:

s51, adding a NaCl solution into the flocculent precipitate until the flocculent precipitate is completely dissolved, centrifuging, discarding the impurity precipitate, and keeping a liquid phase after centrifuging;

s52, adding ethanol into the liquid phase after centrifugation until the precipitation is complete;

repeating S51-S52 until the flocculent precipitate is dissolved in the saturated NaCl solution and no insoluble impurities exist, which indicates that the washing is finished.

The activated sludge contains abundant microbial cells, and endogenous substances contained in the microbial cells need to be released during extraction, and the activated sludge microbial cells are efficiently broken by the method of the steps S11-S13, so that internal substances of the microbial cells are released and enter the muddy water mixed solution; the method is efficient and energy-saving, and is suitable for the treatment of algal polysaccharide wall breaking and releasing.

In addition, in addition to the algal polysaccharides to be extracted, the liquid phase of the wall-broken muddy water mixed liquor contains a plurality of soluble substances (pollutants in the sewage, organic substances such as proteins and nucleic acids simultaneously released in the microbial cells, sewage, inorganic substances released by the microbes, and the like), so the algal polysaccharides need to be extracted from the multiphase mixed liquor by the operations of the steps S2 to S4.

In addition, the algal polysaccharide directly extracted from the mud-water mixture is a mixture of various components and still contains partial impurities, so the algal polysaccharide is purified through repeated washing in the steps S2-S5.

Compared with the prior art, the invention can achieve the following technical effects:

the method for extracting algal polysaccharide from activated sludge provided by the invention takes the activated sludge depended and generated in the biological sewage treatment process of a sewage treatment plant as a raw material, carries out high-efficiency wall breaking on activated sludge microbial cells to release internal substances of the microbial cells, and optimizes the extraction method to obtain purified algal polysaccharide (comprising alginic acid, fucoidan, carrageenan, heparin and the like). The method for extracting algal polysaccharide from activated sludge provided by the invention opens up a new algal polysaccharide large-scale extraction mode except that algae and animal tissues (gastric mucosa) are taken as raw materials. The method not only can provide a new direction and basis for sludge recycling, but also has industrial potential to relieve the problem of single production mode of the current algal polysaccharide.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a flow chart of a method for extracting algal polysaccharides from activated sludge according to an embodiment of the present invention;

FIG. 2 is a solid powder of alginic acid obtained in step (6) of example 1;

FIG. 3 is a precipitated algal polysaccharide solid powder obtained in step (10) of example 1;

FIG. 4 is a diagram of the HPLC analysis of purified algal polysaccharides extracted in example 1;

FIG. 5 is a gel chromatography of purified algal polysaccharide extracted in example 1.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Referring to fig. 1, embodiment 1 of the present invention provides a method for extracting algal polysaccharides from activated sludge, which comprises the following specific steps:

(1) taking 500g (the water content is about 98%) of secondary sedimentation tank concentrated sludge of a certain municipal sewage treatment plant, and adding distilled water until the suspended solid concentration (MLSS) of the concentrated mixed liquor of the activated sludge is 5000 mg/L;

(2) centrifuging at 4500rpm, removing upper liquid phase, adding distilled water with the same volume into the solid-phase mud cake, shaking, centrifuging again, removing upper liquid phase, repeating the mud washing step for 4 times, centrifuging, and retaining the solid-phase mud cake;

(3) adding distilled water into the mud cakes according to a mud-water ratio of 1:5(V/V), uniformly shaking to obtain a mixed solution, heating the mixed solution to 50 ℃, and simultaneously introducing 10V direct current into the mixed solution for 15 minutes according to a period of 'electrifying for 10 seconds, intermittence for 5 seconds, and electrifying for 10 seconds again';

(4) centrifuging at 4500rpm, and retaining upper liquid phase;

(5) carrying out ultrafiltration on the liquid phase component obtained in the step (4) until the volume of the liquid phase is concentrated to 60 percent before ultrafiltration;

(6) adding calcium chloride into the liquid phase after ultrafiltration concentration until the precipitate is complete, centrifuging at 4500rpm, separating the obtained precipitate and the upper liquid phase, collecting the precipitate, and drying to obtain solid alginic acid;

(7) adding 98% ethanol into the upper layer liquid phase in the step (6) until the mass concentration of the ethanol in the solution is 60%, standing at 0 ℃ for 2 hours, centrifuging (4500rpm) after the flocculent precipitate is complete, discarding the supernatant, and keeping the flocculent precipitate;

(8) adding a saturated NaCl solution into the flocculent precipitate obtained in the step (7) until the precipitate is dissolved; centrifuging at 8000rpm, discarding impurity precipitate, and retaining centrifuged liquid phase

(9) Adding 98% ethanol into the liquid phase obtained in the step (8) until the precipitation is complete;

(10) repeating the steps (8) and (9) until saturated NaCl is added into the precipitate and no other insoluble impurities are left after complete dissolution, adding 98% ethanol into the dissolved solution until complete precipitation, and centrifuging at 8000rpm to obtain algal polysaccharide precipitate;

(11) adding distilled water into the algal polysaccharide precipitate until the algal polysaccharide precipitate is completely dissolved, separating solution components by using a space exclusion chromatography, comparing the peak-off time of the algal polysaccharide, the carrageenan and the heparin sodium under the same operation condition, collecting liquid phase separation components, and drying the collected 3 liquid phase components to respectively obtain the algal polysaccharide, the carrageenan and the heparin sodium solid.

The solid powder of alginic acid obtained in step (6) is shown in FIG. 2.

The dried solid powder of the algal polysaccharide precipitate obtained in step (10) is shown in fig. 3, and the algal polysaccharide precipitate is a mixture of brown algae polysaccharide, carrageenan and heparin sodium solid.

Respectively performing ultra performance liquid chromatography analysis and gel chromatography analysis on the algal polysaccharides obtained in the step (10), wherein the specific components and results are respectively shown in fig. 4 and fig. 5. As can be seen from the figure, the algal polysaccharides obtained by the method for extracting algal polysaccharides from activated sludge according to the embodiment of the present invention include alginic acid, fucoidan, carrageenan and heparin, and the total amount is 29% of the dry weight of the sludge, wherein the alginic acid accounts for 8.6% of the dry weight of the sludge, the fucoidan accounts for 5.2% of the dry weight of the sludge, the carrageenan accounts for 12.4% of the dry weight of the sludge, and the heparin accounts for 2.8% of the dry weight of the sludge, and the product purity is high, and the method has an industrial value.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.