CN102977181A - Method for extracting protein in biological sludge - Google Patents

Abstract

The invention discloses a method for extracting protein in biological sludge. The method mainly comprises the steps of: sludge acid-washing, high-temperature acid lysis, isoelectric point determination, isoelectric point crystallization, organic solvent washing and low-temperature drying. A protein solid obtained by separation is fine light yellow powder, and the purity of the protein, which is monitored by adopting an ultraviolet spectrophotometry method with bovine serum albumin as a standard, can be more than 99.8 percent. The infrared spectroscopy of a product proves that the solid powder has remarkable protein structure characteristics. The X-ray fluorometric analysis of the product proves that the protein obtained by separation has no heavy metals of mercury, cadmium and chromium or a toxic element of arsenic and only contains a small amount of lead with the mass content of less than 0.03 percent in the terms of PbO, thereby according with the requirement of a protein feed for the index. A method for separating and recycling protein in sludge generated in the sewage biological treatment process is beneficial to recycling of municipal sludge.

Description

Protein extracting method in the biological sludge

Technical field

Protein extracting method in the biological sludge that the present invention relates to produce in a kind of municipal wastewater biology treatment process belongs to solid waste disposal and resource utilization field.

Background technology

Along with the quickening of socioeconomic development and urbanization process, town sewage and amount of sludge thereof increase fast.Explicitly pointing out the sewage discharge chemical oxygen demand (COD) in China's Eleventh Five-Year Plan report reduces discharging and will reduce discharging 10% on " 15 " basis.Therefore, in recent years, China's Sewage Plant quantity is increased very fast, and the consequent is a large amount of biological sludge.Biological sludge is take microorganism as main body, and is right with the cotton-shaped mixture that suspended substance, colloidalmaterial in the sewage form, and wherein contains a large amount of protein resources.Gross protein value can reach more than 40% in the bibliographical information excess sludge, and therefore, its protein resource has higher utility value.At present, the purposes for sludge protein matter mainly contains following a few class: as animal-feed, as the raw material of frothextinguisher, or prepare biological adhesive.Obviously, in these several purposes, the market outlook of animal-feed are more wide.But as animal-feed strict forage standard is arranged, particularly for aspects such as heavy metal arsenic, lead, cadmium, mercury and chromium comparatively strict content requirement is arranged.It is alkaline process that the sludge protein matter of present bibliographical information shifts to an earlier date method more.The method is conducive to molten born of the same parents' broken wall of mud, discharges protein wherein, but the greatest problem that the method exists is: exist comparatively serious virtue to draw reaction, produce more side reaction, and the extracting solution color is black and glue, and be unfavorable for the separation of protein, and its protein quality is relatively poor.Acid system holds born of the same parents and then can avoid virtue to draw reaction, and the extracting solution color is comparatively limpid, and protein quality is better.But there is the leaching ability of heavy metal problem in traditional acid system direct extraction method, makes easily that heavy metal ion exceeds standard in the proteinaceous product.Therefore, need to develop a kind of new bio sludge protein matter shifts to an earlier date and separating technology.

Summary of the invention

The purpose of this invention is to provide that proteins extraction is in separating technology in the biological sludge in a kind of town sewage biological treatment process, the processing and the resource utilization that can be mud provide the choice of technology.

Technical scheme of the present invention is as follows:

Protein extracting method in a kind of biological sludge, step is as follows:

1) pickling: the biological sludge of dehydration adds water, then adds concentrated hydrochloric acid, makes water ratio and concentrated hydrochloric acid content be respectively 96% and 10%, stirs subsequently 30min, carries out centrifugation again, and making moisture percentage in sewage sludge is 92%;

2) the molten born of the same parents of high-temperature acid: the mud after the first step processed is put into container, then puts into the High-temperature Digestion device, places 2h～8h after being heated to 120 ℃, then carries out centrifugal treating, obtains centrifugal solution;

3) determining of iso-electric point: get at least 6 parts of the solution that second step obtains, every part of 10ml, regulate a series of pH with sodium hydroxide solution, place 12h under the room temperature, carry out subsequently high speed centrifugation and separate, and the pH of the supernatant liquor of measuring, then adopt Folin-phenol reagent process (Lowry method) to measure protein content in the supernatant liquor, take pH as X-coordinate, protein concn is the ordinate zou mapping, minimum corresponding pH is defined as its iso-electric point pI with protein concn;

4) isoelectric point crystallizing: the centrifugal solution remainder that second step is obtained with sodium hydroxide solution is titrated to definite isoelectric pH of the 3rd step, separate out protein solid, place 4h, then adopt centrifugation isolated protein solid, with 95% ethanol this solid is carried out agitator treating again, again carry out centrifugation after the washing and obtain protein solid;

5) drying: the solid that obtains is carried out drying in baking oven, drying temperature is 45 ℃.

Be described as follows:

Isoelectric points of proteins is determined: at least 6 parts of molten born of the same parents' extracting solutions getting every part of 10ml, adopt sodium hydroxide to regulate a series of different pH values, the pH scope is that solid begins to occur to solid and begins to reduce, and placement 12h, then above-mentioned many parts of styles are carried out centrifugation, adopt spectrophotometry to measure protein content at the characteristic wavelength 280nm place of protein to supernatant liquor, and measure the pH of corresponding supernatant liquor, subsequently take pH as X-coordinate, protein content is ordinate zou mapping, according to data determine protein content a hour corresponding pH be the extraction isoelectric point of protein.

Isoelectric points of proteins is separated out: under normal temperature condition, according to the isoelectric points of proteins data that experiment is determined, adopt the pH of sodium hydrate regulator solution to iso-electric point, this moment, protein was separated out, and carried out subsequently centrifugation, obtained protein solid.Adopt subsequently 95% ethanol to wash, and the recentrifuge separation obtain protein.

Protein solid is dry: protein is Biological resources, and its drying temperature can not be too high, otherwise the easy sex change of protein reduces the availability of protein greatly.The protein drying temperature is 45 ℃ in this technique.

Adding method about water and concentrated hydrochloric acid is " adding concentrated hydrochloric acid after adding first entry " again, and 96% water ratio comprises all moisture in the mixed system, and the mass content of concentrated hydrochloric acid refers to concentrated hydrochloric acid mass content in mixture of about 38%;

For step 2) " being heated to 120 ℃ " determine according to process of the test mesohigh digesting equipment self top temperature.Because the molten born of the same parents' process of acid system is slower at low temperatures, therefore molten born of the same parents' experimental installation has adopted and has had the equipment that can be heated to 120 ℃ under the certain pressure and carry out the molten born of the same parents of hot method acid.Therefore, mainly be to regulate its molten born of the same parents' effect heat-up time by changing.

Need to get the molten cytosol of part in definite process of iso-electric point, by regulating different pH, separate out and obtain under the different pH remaining protein concentration in the solution after the protein matter, determine that by protein concn and pH relation the minimum corresponding pH of protein concn is as isoelectric point of protein.Certainly, more many accurately the determining of iso-electric point that more are conducive to of filtrate umber in determining the iso-electric point process, experiment shows that adopting 6 parts can determine iso-electric point.The definite of iso-electric point only needs the single job process to get final product in this research, and iso-electric point all adopts these data in all the other experiments.

" placing 4 hours " in the isoelectric point crystallizing process is storage period minimum in the research, can be longer.In theory storage period longer, the amount that protein solid is separated out should be more some more, but the time is oversize, are unfavorable for operation, then the increase of the amount of separating out of solid is also not obvious, therefore there is no need oversize storage period.

Cleaning solvent gets final product with 95% ethanol, if want high with " dehydrated alcohol " its price, and " 95% ethanol " is regular commodity, and will hang down with respect to the dehydrated alcohol price.

Proteins extraction technique comprises the links such as pickling, centrifugation, washing, drying in the biological sludge provided by the present invention.Acetone or alcohol is adopted in washing.

The invention provides proteins extraction and separation method in a kind of new biological sludge, heavy metal content meets national forage standard in its proteinaceous product.The proteinaceous product purity 102%(that this technique obtains is take bovine serum protein as standard).

Description of drawings

Fig. 1 extracting solution of protein iso-electric point is determined curve.

Proteinaceous product stereoscan photograph among Fig. 2 embodiment 1.

The infrared spectrum of Fig. 3 proteinaceous product.

The x-ray fluorescence analysis collection of illustrative plates of Fig. 4 proteinaceous product.

Embodiment

Embodiment 1.

Get certain Sewage Plant dewatered sludge 500g(water ratio 80%), add distilled water 1750g and concentrated hydrochloric acid 250g, stir at normal temperatures subsequently 30min, and carry out centrifuging, leaching filtrate to water ratio is 92%, then, mud is put into container, put into again the High-temperature Digestion device, at 120 ℃, heating 2h carries out centrifuging after the cooling, obtain the about 450ml of filtrate, all the other filtrates of taking-up filtrate 60ml(are put into refrigerator and are preserved), be equally divided into 6 parts, put into separately the 50ml small beaker, regulate a series of pH with sodium hydroxide solution, be respectively 3.0,4.2,5.2,5.6,6.0 with 7.0, place 12h under the room temperature, carry out subsequently high speed centrifugation and separate, keep supernatant liquor, and measure the pH of above-mentioned corresponding supernatant liquor, be respectively 2.9,3.6,4.7,5.3,5.6 with 6.5, then adopt Fei Lin-phenol method to measure protein content (take bovine serum protein as standard) in the supernatant liquor, take pH as X-coordinate, protein concn is ordinate zou mapping (seeing accompanying drawing 1), determines that its iso-electric point pI is 5.3.Subsequently to residual filtrate, sodium hydroxide with 10% is regulated pH to 5.3, normal temperature is placed 4h, carry out subsequently centrifugation, with 95% ethanol solid is washed, carrying out centrifugation subsequently, the solid that obtains is put into the desiccated surface ware, put into 45 ℃ of baking ovens and carry out drying, weighing after the drying obtains faint yellow solid material 7.02g.The mass content of measuring its protein with Fei Lin-phenol method is that 103.8%(is take bovine serum albumin as monitoring standard).Infared spectrum as shown in Figure 3.3406.31cm-1 is N-H stretching vibration in the protein peptide bond in the infrared spectrum, 2967.15cm-1 be the stretching vibration of c h bond, 1656.65cm-1 be the C=O stretching vibration in the protein peptide bond, acid amides I bands of a spectrum, 1551.16cm-1 in the protein peptide bond-N-H bond bending vibration among the CONH-and the stretching vibration of C-N key cause acid amides II bands of a spectrum.The x-ray fluorescence analysis collection of illustrative plates as shown in Figure 4.Heavy metal chromium, cadmium, mercury and arsenic element all do not detect, plumbous (take PbO) mass content in the element that can monitor (ordination number is greater than 11) is 0.02%, therefore, the protein of the method extraction meets the quality standard of heavy metal and the arsenic of protein feed fully.

Embodiment 2.

Identical with embodiment 1, only be to be respectively 5h and 8h heat-up time, and need not to determine again isoelectric points of proteins, the filtrate that obtains is directly carried out protein and is separated out under the iso-electric point that embodiment 1 determines, adopt subsequently 95% ethanol to wash, and carrying out drying, the solid matter that obtains is respectively 14.43g and 16.76g.Its protein content is respectively 102.1% and 99.8%.Corresponding heavy metal chromium, cadmium, mercury and arsenic all do not detect, and plumbous (in PbO) mass content is respectively 0.02% and 0.03%, meets protein feed to the quality standard-required.

The present invention open and propose biological sludge in protein extracting method, be a kind of easy, the measuring method fast and accurately of suspended particles concentration determination in the draining in the biological sewage treatment.Those skilled in the art can be by using for reference this paper content, and the preparation method of appropriate change mud suspension liquid concentration or detection target can realize.Special needs to be pointed out is, the replacement that all are similar and change apparent to those skilled in the artly, they are deemed to be included in spirit of the present invention, scope and the content.

Claims (1)

1. protein extracting method in the biological sludge is characterized in that step is as follows:

1) pickling: the biological sludge of dehydration adds water, then adds concentrated hydrochloric acid, makes water ratio and concentrated hydrochloric acid content be respectively 96% and 10%, stirs subsequently 30min, carries out centrifugation again, and making moisture percentage in sewage sludge is 92%;

2) the molten born of the same parents of high-temperature acid: the mud after the first step processed is put into container, then puts into the High-temperature Digestion device, places 2h～8h after being heated to 120 ℃, then carries out centrifugal treating, obtains centrifugal solution;

3) determining of iso-electric point: get at least 6 parts of the solution that second step obtains, every part of 10ml, regulate a series of pH with sodium hydroxide solution, place 12h under the room temperature, carry out subsequently high speed centrifugation and separate, and the pH of the supernatant liquor of measuring, then adopt Fei Lin-phenol method to measure protein content in the supernatant liquor, take pH as X-coordinate, protein concn is the ordinate zou mapping, minimum corresponding pH is defined as its iso-electric point pI with protein concn;

4) isoelectric point crystallizing: the centrifugal solution remainder that second step is obtained with sodium hydroxide solution is titrated to definite isoelectric pH of the 3rd step, separate out protein solid, place 4h, then adopt centrifugation isolated protein solid, with 95% ethanol this solid is carried out agitator treating again, again carry out centrifugation after the washing and obtain protein solid;

5) drying: the solid that obtains is carried out drying in baking oven, drying temperature is 45 ℃.

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