CN113897318A - Method for co-culturing protein degradation by using acetoacidophilic protein bacterium and sulfur reducing geobacillus - Google Patents
Method for co-culturing protein degradation by using acetoacidophilic protein bacterium and sulfur reducing geobacillus Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 44
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000012258 culturing Methods 0.000 title claims abstract description 15
- 241000626621 Geobacillus Species 0.000 title claims abstract description 13
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 title claims description 7
- 229910052717 sulfur Inorganic materials 0.000 title claims description 7
- 239000011593 sulfur Substances 0.000 title claims description 7
- 230000017854 proteolysis Effects 0.000 title abstract description 22
- 238000011081 inoculation Methods 0.000 claims abstract description 26
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- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 4
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 4
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 claims description 4
- GNHOJBNSNUXZQA-UHFFFAOYSA-J potassium aluminium sulfate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GNHOJBNSNUXZQA-UHFFFAOYSA-J 0.000 claims description 4
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 4
- 229960002477 riboflavin Drugs 0.000 claims description 4
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- 239000011684 sodium molybdate Substances 0.000 claims description 4
- 235000015393 sodium molybdate Nutrition 0.000 claims description 4
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 4
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
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- 241000589220 Acetobacter Species 0.000 claims 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 42
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for co-culturing and degrading protein by using acetoacidophilic protein bacteria and sulfur-reducing geobacillus, which comprises the following specific steps: s1, inoculating acetoacidophilic proteophile and thioredoxin in the culture solution; s2 protein degradation effect: the degradation effect of the protein of the mixed bacteria at different days and different inoculation ratios is observed, and the result shows that: the co-culture of the two bacteria is far superior to the degradation effect of pure culture of the acetoacidophilic protein bacteria on protein; the degraded acetoacidophilic protein bacterium is a protein degradation bacterium and can generate acetic acid, the acetic acid can be used as a substrate of the thioreducing Bacillus, compared with other protein degradation bacteria, the mixed system can degrade more proteins, and the operation is simple and convenient.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for co-culturing and degrading proteins by using acetoacidophilic protein bacteria and sulfur-reducing geobacillus.
Background
Acetoacidophilic protein bacteria (proteoliophiles TB107T) are gram-negative and anaerobic protein-degrading bacteria, which were first isolated from granular sludge from the treatment of beer wastewater and are capable of degrading yeast extract, peptone, pyruvic acid and other complex substrates to produce volatile fatty acid acetic acid and the like, and thiobacillus thioredoxin (Geobacter sulfurlowers PCA) is also an anaerobic bacteria capable of using acetic acid, propionic acid and the like as substrates.
As the number of sewage treatment plants is increased on a large scale, a large amount of domestic sewage and a large amount of sludge are generated, the sewage and the sludge contain a large amount of protein besides a large amount of pollutants, the sewage is rich in a large amount of protein resources, amino acids and growth factors, due to the difficulty in resource recovery, the random discharge of high-protein sewage wastes resources and certain damage to the ecological environment, and therefore, the full recovery and degradation of the resources are urgently needed.
The existing methods for recovering and degrading proteins in sewage and sludge include protein extraction, degradation and differentiation treatment, and direct protein degradation in sewage and sludge, such as thermal acid hydrolysis, thermal alkali hydrolysis and wet oxygen hydrolysis in chemical hydrolysis; ultrasonic methods among physical methods; biological processes such as enzymatic hydrolysis; a combined method such as an ultrasonic combined enzyme method, an ultrasonic combined acid method and the like, for example, Chinese patent document CN109400671A adopts an alkaline low-temperature hydrolysis method to treat sludge, break biological cell walls, release protein, and adopts macroporous exchange resin to refine filtrate so as to obtain a higher degradable protein solution; however, these methods are complicated, expensive, unstable in treatment effect, long in running time, and only remove them, thereby wasting a lot of valuable nutrients.
Disclosure of Invention
The present invention aims at providing the method for co-culturing the acetoacidophilic protein-producing bacteria and the sulfur-reducing bacillus to degrade the protein, so as to solve the problems in the background technology.
In order to solve the technical problems, the invention provides the following technical scheme:
the method comprises the following steps:
s1, inoculating acetoacidophilic proteophile and thioredoxin in the culture solution;
s2: and (3) taking the bacterial liquid, centrifuging at a high speed for 8min, filtering the centrifuged supernatant through a filter membrane, adding the BCA working solution and water into the filtered supernatant, standing at 37 ℃ for 20min, and measuring the absorbance of the sample at 562 nm.
Further, the preparation method of the culture solution comprises the following steps:
a: adding polypeptone, yeast extract, tryptone, glucose, ammonium chloride, calcium chloride, potassium chloride, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, vitamin solution and trace element solution into water;
b: heating in water bath until dissolving, and adjusting pH to 6.9-7.0 with sodium hydroxide solution;
c: introducing mixed gas of nitrogen and hydrogen, aerating for 20min, sterilizing at 121 deg.C for 20min, and ultraviolet irradiating to obtain culture solution.
Further, the inoculation ratio of the protein acetophilic bacteria to the Acinetobacter thioredoxin in the step S1 is (1-4): (1-8).
Further, the inoculation time interval of the acetoacidophilic protein bacteria and the sulfur reducing bacteria in the step S1 is 0-3 days.
5. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 1, wherein: the inoculation amount of the mixed bacterium liquid of the acetoacidophilic proteophile and the sulfur reducing geobacillus is 3-6%, the culture temperature is 34 ℃, and the culture time is 7-9 days.
Further, the materials required for the culture solution include: 2.5-3g/L of polypeptone, 5.0-6g/L of yeast extract, 2.5-3g/L of tryptone, 10.0-12g/L of glucose, 1.5-3g/L of ammonium chloride, 0.024-0.2g/L of calcium chloride, 0.1-0.3g/L of potassium chloride, 0.64-0.8g/L of sodium dihydrogen phosphate, 0.04-0.1g/L of dipotassium hydrogen phosphate, 5-8mL of vitamin solution, 5-8mL of trace element solution and water with constant volume of 1L.
Further, the vitamin solution comprises the following materials: 2-5mg of biotin, 2-5mg of folic acid, 10-15mg of vitamin B6 pyridoxine-hydrochloric acid, 5-8mg of thiamine-hydrochloric acid, 5-8mg of riboflavin, 35-8mg of vitamin B, 5-8mg of D-calcium pantothenate, 120.1-0.3 mg of vitamin B, 5-8mg of p-aminobenzoic acid and 5-8mg of lipoic acid, and the volume of water is fixed to 1L.
Further, the materials required by the trace element liquid comprise: 1.5-3g of nitrilotriacetic acid, 3-5g of magnesium sulfate heptahydrate, 0.5-1g of manganese sulfate monohydrate, 1-3g of sodium chloride, 0.1-0.3g of ferrous sulfate heptahydrate, 0.1-0.3g of cobalt chloride hexahydrate, 0.76-1g of calcium chloride, 0.1-0.3g of zinc sulfate heptahydrate, 0.01-0.05g of copper sulfate pentahydrate, 0.02-0.05g of aluminum potassium sulfate dodecahydrate, 0.01-0.03g of boric acid, 0.01-0.03g of sodium molybdate, and the volume of water is up to 1L.
Further, the acetoacidophilic proteobacteria belong to the genus proteus species.
Further, the thioreductioned geobacter is a strain belonging to the genus geobacter.
Compared with the prior art, the invention has the following beneficial effects: firstly, the protein degradation of the microorganism can be carried out under mild conditions, and the defects of pollution, high energy consumption and complex operation caused by protein treatment by a physical and chemical method can be avoided.
Secondly, the strains used in the invention all belong to anaerobic mode bacteria, and can better degrade protein compared with other anaerobic bacteria, and the operation is simple and convenient because an anaerobic incubator is not required to be degraded.
Thirdly, the mixed bacteria promote the degradation of protein under the anaerobic condition, the efficiency is high, the period is short, the sulfur-reducing geobacillus can utilize the generated acetic acid under the condition that the pure bacteria of the acetotropic bacteria can degrade the protein, thereby reducing the adverse effect of product accumulation on the growth of bacteria, opening up a new way for the treatment of the protein in the sewage, and further providing a new way for the resource treatment of the protein wastewater.
Fourthly, the influence of the inoculation sequence and time of the bacterial liquid on the degradation of the protein is researched, firstly, the acetoacidophilic proteus can convert the protein into acetic acid, and the sulfur-reducing geobacillus can take acetic acid molecules as substrates to carry out self growth and metabolism, so that the acetoacidophilic proteus is inoculated for self growth and metabolism, then the sulfur-reducing geobacillus is inoculated, and meanwhile, orthogonal experiments show that the acetoacidophilic proteus and the sulfur-reducing geobacillus are inoculated simultaneously, and the degradation effect of the protein is better than that of the acetoacidophilic proteus for 2-3 days and then the sulfur-reducing geobacillus.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the protein degradation of examples A1-A4 and comparative example 1;
FIG. 2 shows the protein degradation of example B1-B4 and comparative example 2;
FIG. 3 shows the growth of the mixed bacterial liquid of examples B1-B4 and comparative example 2;
FIG. 4 shows the use of acetic acid in the mixed bacterial liquid of examples B1-B4 and comparative example 2.
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 of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
Taking example a1 as an example:
s1 Co-culture of two bacteria:
a: preparing a culture solution: adding 500mL of distilled water into a 1L triangular flask, and dividingRespectively adding polypeptone 2.5g/L, yeast extract 5.0g/L, tryptone 2.5g/L, glucose 10.0g/L, ammonium chloride 1.5g/L, calcium chloride 0.024g/L, potassium chloride 0.1g/L, sodium dihydrogen phosphate 0.64g/L, dipotassium hydrogen phosphate 0.04g/L, vitamin solution 5mL, and trace element solution 5mL, adding pure water to constant volume to 1L, heating in water bath to dissolve, adding NaOH dropwise to adjust pH to 6.9, filling into a bottle, inserting aeration needle into the bottle, introducing 80% N, adding sodium hydroxide, sodium hydroxide2And 20% of H2Aerating the mixed gas for 20min, quickly covering the rubber stopper, placing the narrow-mouth bottle in an autoclave, sterilizing at 121 ℃ for 20min, and then transferring to a super clean bench for ultraviolet irradiation for later use;
b: inoculation: inoculating acetoacidophilic proteophile and then thioredoxin, wherein the inoculation ratio of the acetoacidophilic proteophile to the thioredoxin is 1:4, and the two are co-cultured for 8 days, and the inoculation amount is 5%;
s2: protein degradation effect:
taking 8 days as a period, taking 1mL of bacterial liquid at the same time every day, centrifuging for 8min at 10000r/min, filtering the supernatant obtained after centrifugation through a PES filter membrane of 0.22 mu m, taking 200 mu L of filtered supernatant 20 mu L, BCA working solution (BCA protein concentration determination kit produced by Fuzhou Feijing Biotechnology Co., Ltd.), adding water to fix the volume to 2.2mL, placing the prepared sample at 37 ℃ for 20min, measuring the absorbance of the sample at the wavelength of 562nm by using an ultraviolet-visible spectrophotometer, and taking the culture solution of the non-inoculated acetogenium and the thioredoxin as a blank group.
The preparation method of the vitamin solution comprises the following steps: mixing biotin 2.00mg, folic acid 2.00mg, vitamin B6 pyridoxine-hydrochloric acid 10.00mg, thiamine-hydrochloric acid 5.00mg, riboflavin 5.00mg, and vitamin B35.00mg, 5.00mg of calcium D-pantothenate, vitamin B120.100mg, 5.00mg of p-aminobenzoic acid and 5.00mg of lipoic acid are uniformly mixed, and then the mixture is added with water to be constant volume to 1L.
The preparation method of the trace element liquid comprises the following steps: 1.50g of nitrilotriacetic acid, 3.00g of magnesium sulfate heptahydrate, 0.50g of manganese sulfate monohydrate, 1.00g of sodium chloride, 0.10g of ferrous sulfate heptahydrate, 0.10g of cobalt chloride hexahydrate, 0.76g of calcium chloride, 0.10g of zinc sulfate heptahydrate, 0.01g of copper sulfate pentahydrate, 0.02g of aluminum potassium sulfate dodecahydrate, 0.01g of boric acid and 0.01g of sodium molybdate are uniformly mixed, and then water is used for fixing the volume to 1L.
Examples A1-A4: the specific data obtained by changing the inoculation time in example A1 are shown in Table 1, and the specific data obtained by measuring the protein degradation rate of the mixed bacterial liquid are shown in Table 2.
TABLE 1 example A1-A4 Mixed bacterial liquid inoculation time
| A1 | A2 | A3 | A4 | |
| Time of |
0 | 1 | 2 | 3 |
Comparative example 1: in contrast to example 1, the culture broth was inoculated with a Propionibacterium acetogenium.
TABLE 2 Effect of different inoculation times on protein degradation rates in examples A1-A4
Taking example B1 as an example:
s1 Co-culture of two bacteria:
a: preparing a culture solution: adding 500mL of distilled water into a 1L triangular flask, respectively adding polypeptone 2.5g/L, yeast extract 5.0g/L, tryptone 2.5g/L, glucose 10.0g/L, ammonium chloride 1.5g/L, calcium chloride 0.024g/L, potassium chloride 0.1g/L, sodium dihydrogen phosphate 0.64g/L, dipotassium hydrogen phosphate 0.04g/L, vitamin solution 5mL and microelement solution 5mL, adding pure water to a constant volume of 1L, heating in water bath to dissolve, dropwise adding NaOH to adjust pH to 7.0, packaging into a narrow-mouth bottle, inserting an aeration needle into the narrow-mouth bottle, introducing 80% N2And 20% of H2Aerating the mixed gas for 20min, quickly covering the rubber stopper, placing the narrow-mouth bottle in an autoclave, sterilizing at 121 ℃ for 20min, and then transferring to a super clean bench for ultraviolet irradiation for later use;
b: inoculation: inoculating acetoacidophilic proteophile, inoculating thioredoxin after 2 days, and co-culturing for 8 days with the inoculation amount of 5%;
s2: protein degradation effect:
taking 8 days as a period, taking 1mL of bacterial liquid at the same time every day, centrifuging for 8min at 10000r/min, filtering the supernatant obtained after centrifugation through a PES filter membrane of 0.22 mu m, taking 200 mu L of filtered supernatant 20 mu L, BCA working solution (BCA protein concentration determination kit produced by Fuzhou Feijing Biotechnology Co., Ltd.), adding water to fix the volume to 2.2mL, placing the prepared sample at 37 ℃ for 20min, measuring the absorbance of the sample at the wavelength of 562nm by using an ultraviolet-visible spectrophotometer, and taking the culture solution of the non-inoculated acetogenium and the thioredoxin as a blank group.
The preparation method of the vitamin solution comprises the following steps: 2.00mg of biotin, 2.00mg of folic acid, 10.00mg of vitamin B6 pyridoxine-hydrochloric acid, 5.00mg of thiamine-hydrochloric acid, 5.00mg of riboflavin, 35.00mg of vitamin B, 5.00mg of D-calcium pantothenate, 120.100mg of vitamin B, 5.00mg of p-aminobenzoic acid and 5.00mg of lipoic acid are uniformly mixed, and then the volume is fixed to 1L by water.
The preparation method of the trace element liquid comprises the following steps: 1.50g of nitrilotriacetic acid, 3.00g of magnesium sulfate heptahydrate, 0.50g of manganese sulfate monohydrate, 1.00g of sodium chloride, 0.10g of ferrous sulfate heptahydrate, 0.10g of cobalt chloride hexahydrate, 0.76g of calcium chloride, 0.10g of zinc sulfate heptahydrate, 0.01g of copper sulfate pentahydrate, 0.02g of aluminum potassium sulfate dodecahydrate, 0.01g of boric acid and 0.01g of sodium molybdate are uniformly mixed, and then water is used for fixing the volume to 1L.
Examples B1-B4: the specific data obtained by replacing the inoculation ratio in example B1 are shown in Table 3, and the specific data obtained by measuring the protein degradation rate of the mixed bacterial liquid are shown in Table 4.
TABLE 3 example B1-B4 inoculation ratio of mixed bacterial liquid
Comparative example 2: in contrast to example 2, the culture broth was inoculated with a Propionibacterium acetogenium.
TABLE 4 Effect of different inoculation ratios on protein degradation rates in examples B1-B4
Experimental data
Mixed culture growth (OD 600): 2mL of the culture solution was taken, and the absorbance (blank was a culture solution of non-inoculated bacteria) was measured at 600nm once a day.
Acetic acid utilization: centrifuging 1mL of bacterial liquid at 10000r/min and room temperature for 8min, filtering the supernatant obtained after centrifugation through a PES filter membrane of 0.22 mu m, adding 0.9mL of methanol into 0.1mL of the filtered supernatant, centrifuging at 10000r/min and room temperature for 5min, adding 0.15mL of the supernatant into pure water to dilute the pure water by 10 times, and performing analysis and detection on the obtained solution, namely a sample solution, in an ion chromatography, wherein a culture solution of non-inoculated bacteria is used as a blank, and the blank solution is treated in the same way as the sample.
TABLE 5 Effect of different inoculation times on protein degradation rates in examples A1-A4
TABLE 6 Effect of different inoculation ratios on protein degradation rates in examples B1-B4
TABLE 7 results of experiments in examples B1-B4 and comparative example 2OD600
| B1 | B2 | B3 | B4 | Comparative example 2 | |
| |
0 | 0 | 0 | 0 | 0 |
| |
0 | 0 | 0 | 0 | 0 |
| 2d | 0.078 | 0.078 | 0.078 | 0.078 | 0.06 |
| 3d | 0.268 | 0.281 | 0.36 | 0.282 | 0.139 |
| 4d | 0.302 | 0.407 | 0.361 | 0.285 | 0.232 |
| 5d | 0.277 | 0.371 | 0.327 | 0.277 | 0.275 |
| 6d | 0.272 | 0.371 | 0.319 | 0.277 | 0.254 |
| 7d | 0.262 | 0.323 | 0.327 | 0.251 | 0.251 |
| 8d | 0.270 | 0.338 | 0.274 | 0.312 | 0.217 |
TABLE 8 utilization of acetic acid in examples B1-B4 and comparative example 2
And (4) conclusion: as can be seen from tables 2 and 4, the protein degradation rate of the sulfur-reducing Bacillus strain inoculated at different inoculation times and different inoculation ratios is higher than that of the control group, the acetoacidophilic Propionibacterium is inoculated for 2 days and then the thioreducing Bacillus strain is inoculated, the protein degradation rate is highest when the inoculation ratio is 1:4, the protein degradation rate is accelerated after the thioreducing Bacillus strain is inoculated, the mixed culture is more favorable for the degradation of the protein, Table 7 shows that the mixed growth condition is best when the biomass of the mixed culture is higher than that of the comparative example 2 and 1:4, the bacterial growth condition and the protein degradation rate are in a positive relationship, Table 8 shows that the acetic acid content of the comparative example 2 is in an ascending trend and is finally higher than that of the mixed strain, while the acetic acid concentrations under different inoculation ratios in the mixed strain are closer, the acetic acid content is obviously reduced after the thioreducing Bacillus strain is added, the change of acetic acid and the protein degradation rate do not form a positive-negative relationship.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for co-culturing and degrading protein by using acidophilic protein bacteria and sulfur reducing geobacillus is characterized by comprising the following steps: the method comprises the following steps:
s1, inoculating acetoacidophilic proteophile and thioredoxin in the culture solution;
s2: and (3) taking the bacterial liquid, centrifuging at a high speed for 8min, filtering the centrifuged supernatant through a filter membrane, adding the BCA working solution and water into the filtered supernatant, standing at 37 ℃ for 20min, and measuring the absorbance of the sample at 562 nm.
2. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 1, wherein: the preparation method of the culture solution comprises the following steps:
a: adding polypeptone, yeast extract, tryptone, glucose, ammonium chloride, calcium chloride, potassium chloride, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, vitamin solution and trace element solution into water;
b: heating in water bath until dissolving, and adjusting pH to 6.9-7.0 with sodium hydroxide solution;
c: introducing mixed gas of nitrogen and hydrogen, aerating for 20min, sterilizing at 121 deg.C for 20min, and ultraviolet irradiating to obtain culture solution.
3. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 1, wherein: the inoculation ratio of the protein acetophilic bacteria to the Acetobacter thioredoxin in the step S1 is (1-4): (1-8).
4. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 1, wherein: in the step S1, the inoculation time interval of the acetoacidophilic protein bacteria and the sulfur reducing geobacillus is 0-3 days.
5. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 1, wherein: the inoculation amount of the mixed bacterium liquid of the acetoacidophilic proteophile and the sulfur reducing geobacillus is 3-6%, the culture temperature is 34 ℃, and the culture time is 7-9 days.
6. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 2, wherein: the materials required by the culture solution comprise: 2.5-3g/L of polypeptone, 5.0-6g/L of yeast extract, 2.5-3g/L of tryptone, 10.0-12g/L of glucose, 1.5-3g/L of ammonium chloride, 0.024-0.2g/L of calcium chloride, 0.1-0.3g/L of potassium chloride, 0.64-0.8g/L of sodium dihydrogen phosphate, 0.04-0.1g/L of dipotassium hydrogen phosphate, 5-8mL of vitamin solution, 5-8mL of trace element solution and water with constant volume of 1L.
7. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 2, wherein: the vitamin solution comprises the following materials: 2-5mg of biotin, 2-5mg of folic acid, 10-15mg of vitamin B6 pyridoxine-hydrochloric acid, 5-8mg of thiamine-hydrochloric acid, 5-8mg of riboflavin, and vitamin B35-8mg, 5-8mg of D-calcium pantothenate, vitamin B120.1-0.3mg, 5-8mg of p-aminobenzoic acid and 5-8mg of lipoic acid, and the volume of water is fixed to 1L.
8. The method for degrading proteins by co-culturing the acetoacidophilic bacterium and the thioreductic bacterium according to claim 2, wherein: the materials required by the trace element liquid comprise: 1.5-3g of nitrilotriacetic acid, 3-5g of magnesium sulfate heptahydrate, 0.5-1g of manganese sulfate monohydrate, 1-3g of sodium chloride, 0.1-0.3g of ferrous sulfate heptahydrate, 0.1-0.3g of cobalt chloride hexahydrate, 0.76-1g of calcium chloride, 0.1-0.3g of zinc sulfate heptahydrate, 0.01-0.05g of copper sulfate pentahydrate, 0.02-0.05g of aluminum potassium sulfate dodecahydrate, 0.01-0.03g of boric acid, 0.01-0.03g of sodium molybdate, and the volume of water is up to 1L.
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