CN114438069B - Double microcarriers for culturing nitrifying bacteria and preparation method and application thereof - Google Patents
Double microcarriers for culturing nitrifying bacteria and preparation method and application thereof Download PDFInfo
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- CN114438069B CN114438069B CN202011197027.6A CN202011197027A CN114438069B CN 114438069 B CN114438069 B CN 114438069B CN 202011197027 A CN202011197027 A CN 202011197027A CN 114438069 B CN114438069 B CN 114438069B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 108
- 230000001546 nitrifying effect Effects 0.000 title claims abstract description 70
- 238000012258 culturing Methods 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229920001661 Chitosan Polymers 0.000 claims abstract description 37
- 230000012010 growth Effects 0.000 claims abstract description 17
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 14
- 238000001035 drying Methods 0.000 claims abstract description 10
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 8
- 238000001179 sorption measurement Methods 0.000 claims abstract description 6
- 239000007787 solid Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 25
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 12
- 238000004132 cross linking Methods 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 7
- 239000011575 calcium Substances 0.000 claims description 7
- 229910052791 calcium Inorganic materials 0.000 claims description 7
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 6
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 claims description 6
- 241000186660 Lactobacillus Species 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- 238000012136 culture method Methods 0.000 claims description 6
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 claims description 6
- 229940039696 lactobacillus Drugs 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 238000006477 desulfuration reaction Methods 0.000 claims description 5
- 230000023556 desulfurization Effects 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 241000222178 Candida tropicalis Species 0.000 claims description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
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- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 238000000855 fermentation Methods 0.000 claims description 4
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
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- 239000011159 matrix material Substances 0.000 claims description 3
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- 241000186000 Bifidobacterium Species 0.000 claims description 2
- 241001337994 Cryptococcus <scale insect> Species 0.000 claims description 2
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 2
- 241000194036 Lactococcus Species 0.000 claims description 2
- 241000223252 Rhodotorula Species 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 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
- 238000007385 chemical modification Methods 0.000 claims description 2
- -1 formaldehyde, crown ethers Chemical class 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 230000009977 dual effect Effects 0.000 claims 3
- 241000235342 Saccharomycetes Species 0.000 claims 1
- 230000003698 anagen phase Effects 0.000 claims 1
- 230000001502 supplementing effect Effects 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 9
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- 230000000694 effects Effects 0.000 abstract description 5
- 230000002411 adverse Effects 0.000 abstract description 3
- 239000000969 carrier Substances 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 24
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 239000004005 microsphere Substances 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000008157 edible vegetable oil Substances 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
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- 238000012986 modification Methods 0.000 description 2
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- 229920000642 polymer Polymers 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000010413 sodium alginate Nutrition 0.000 description 2
- 229940005550 sodium alginate Drugs 0.000 description 2
- 239000000661 sodium alginate Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- 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
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Abstract
The invention relates to a double microcarrier for culturing nitrifying bacteria, a preparation method and application thereof, wherein a cross-linked chitosan carrier embedded with calcium carbonate is prepared; and (3) adding the cross-linked chitosan carrier into a heterotrophic bacteria culture system utilizing an organic carbon source for adsorption growth, culturing until the late growth stage is stopped, taking out a solid and drying to obtain the double microcarriers. The double microcarriers provided by the invention can eliminate the adverse effect of chitosan on microorganisms, and are beneficial to the combination of nitrifying bacteria and carriers, so that the culture density of nitrifying bacteria is improved.
Description
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a double microcarrier for culturing nitrifying bacteria, and a preparation method and application thereof.
Background
Nitrifying bacteria belong to aerobic autotrophic microorganisms, and under the condition of sufficient supply, energy is obtained from the process of oxidizing ammonia and nitrite so as to assimilate CO 2 The growth is slow, the generation time is long, and the growth is easily influenced by a plurality of factors such as substrate concentration, dissolved oxygen concentration, pH and the like, so that the proliferation speed is low, the rapid culture is not easy, and the industrial large-scale application is difficult.
Nitrifying bacteria generally grow on the wall, and a large adhesion surface area is provided by using a moving bed biological fluidized bed filler, a biochemical blanket and the like in ornamental fish culture and sewage treatment so as to be beneficial to adhesion and growth of nitrifying bacteria. Compared with the traditional carrier, the microcarrier generally has larger specific surface area and better substrate diffusion effect, and has been widely applied in animal cell suspension culture for early years. Chitosan can be used as a microcarrier, but since chitosan is a polymer with positive charges on the surface and has a certain antibacterial property, the direct use as microcarrier can affect the growth of bacteria.
CN109095623a discloses a microcarrier for improving culture density of aerobic nitrifying bacteria and nitrosating bacteria and a preparation method thereof, which is prepared by adopting the following method: configuration of Na 2 CO 3 Solution and CaCl 2 A solution; respectively adding polyacrylic acid solutions; adding sodium dodecyl sulfate solution into the mixed solution A to prepare mixed solution C, and then pouring the mixed solution B into the mixed solution C to react to generate precipitate; filtering and washing the precipitate, and then drying in vacuum; dissolving chitosan solution in acetic acid solution, adding calcium carbonate nanoparticles, edible oil and span-80, stirring, and adding genipin for continuous stirring; or glutaraldehyde solution is added, and borohydride or acetyl borohydride is continuously added for continuous reaction after stirring; and (3) centrifugally separating the precipitate, cleaning, dehydrating and airing to obtain yellowish powder, namely the calcium carbonate-embedded chitosan microcarrier. Suspending the chitosan microcarrier embedded with calcium carbonate in water, dropwise adding the water into a sodium alginate solution, stirring, centrifugally separating and precipitating, cleaning, soaking the precipitate in a hydrochloric acid solution with the mass concentration of 1-10%, centrifugally separating and cleaning after the treatment, centrifugally separating and precipitating, and finally dehydrating the precipitate by using acetone and airing the dehydrated precipitate at room temperature to obtain the chitosan microcarrier with the surface charge modified, namely the microcarrier for improving the culture density of aerobic nitrifying bacteria and nitrosating bacteria. The microcarrier can improve the culture density of nitrifying bacteria and improve the economy of the enlarged culture of nitrifying bacteria. The method is characterized in that the crosslinked chitosan microsphere is dripped into a negatively charged polymer sodium alginate solution, the crosslinked chitosan is utilized to carry out surface charge modification to prepare the microcarrier, a layer of negatively charged shell is formed on the surface of the microcarrier, and although the influence of the chitosan on the growth of nitrifying bacteria is eliminated, the electrostatic adsorption of the chitosan microsphere to microorganisms is reduced, and the surface charge modification is not beneficial to the adhesion growth of negatively charged nitrifying bacteria on the carrier.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a double microcarrier for culturing nitrifying bacteria, and a preparation method and application thereof. The double microcarriers provided by the invention can eliminate the adverse effect of chitosan on microorganisms, and are beneficial to the combination of nitrifying bacteria and carriers, so that the culture density of nitrifying bacteria is improved.
The invention provides a preparation method of a double microcarrier for culturing nitrifying bacteria, which comprises the following steps:
preparing a cross-linked chitosan carrier embedded with calcium carbonate; and (3) adding the calcium carbonate-embedded cross-linked chitosan carrier into a heterotrophic bacteria culture system utilizing an organic carbon source for adsorption growth, culturing until the late growth stage is stopped, taking out a solid and drying to obtain the double microcarrier.
In the invention, the calcium carbonate-embedded cross-linked chitosan carrier can be obtained by adopting a preparation method conventional in the field. The crosslinking method mainly adopts direct crosslinking, chemical modification in crosslinking and the like. The crosslinking agent used in the direct crosslinking method is at least one of epichlorohydrin, glutaraldehyde, formaldehyde, crown ethers, genipin and the like, and genipin is preferred. The cross-linking is that the cross-linking reaction is carried out between chitosan and the cross-linking agent molecules, so that the chitosan molecules are changed into a net structure from a straight chain, the specific surface area, the pore structure and other physical properties of the chitosan can be improved through the cross-linking, and the stability of the chitosan is effectively improved.
In the present invention, the heterotrophic bacteria may be at least one of the heterotrophic bacteria using an organic carbon source, such as yeast, lactic acid bacteria, and sulfate-reducing bacteria, and preferably yeast. The yeast may be at least one selected from candida, cryptococcus, hansenula, pichia, rhodotorula, torulopsis or candida, and preferably candida tropicalis. The lactobacillus can be at least one selected from lactobacillus, bifidobacterium, lactococcus and the like. The sulfate reducing bacteria can be at least one selected from the group consisting of desulfurization monads, desulfurization lineaes and the like.
In the invention, the organic carbon source is determined according to the specific strain selected, and is generally selected from carbohydrate, protein, organic acid and other carbon-containing organic matters used in conventional culture of heterotrophic bacteria, such as at least one of glucose, hexose, xylose, sucrose, starch and the like. The organic carbon source is added according to the mass concentration of 1-5g/L in the system after the addition.
In the invention, the culture conditions of the heterotrophic bacteria are as follows: the temperature is 20-38deg.C, preferably 20-30deg.C, and pH is 6.0-8.5, preferably 6.0-7.0; and (3) carrying out stationary fermentation or shake culture, wherein stirring is carried out every 30-60min, and the rotation speed of the shake culture is 200-600r/min. Culturing to late logarithmic growth stage is generally carried out for 24-80 h.
In the invention, the drying temperature is 25-50 ℃ and the drying time is 1-5h.
The double microcarrier for culturing nitrifying bacteria is prepared by the method. The prepared double microcarrier takes crosslinked chitosan as a matrix, and heterotrophic bacteria are adsorbed and grown on the double microcarrier, wherein the heterotrophic bacteria account for 5% -50%, preferably 10% -30% of the carrier.
The invention also provides a culture method for improving the culture density of the nitrifying bacteria, and the double microcarriers for culturing the nitrifying bacteria provided by the invention are adopted and added into a nitrifying bacteria enrichment culture system.
In the culture method, the ratio of the adding amount of the double microcarriers to the culture solution in the nitrifying bacteria enrichment culture system is 1-5g/L.
In the culture method, the volume ratio of the adding amount of the nitrifying bacteria to the culture solution in the nitrifying bacteria enrichment culture system is 0.5-5%.
In the culture method, the enrichment culture of nitrifying bacteria can adopt batch water exchange and drainage or batch feed supplement.
In the culture method, the enrichment culture conditions of nitrifying bacteria are as follows: the temperature is 28-35 ℃, the pH is 7.2-8.2, and the dissolved oxygen is 1-5mg/L.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts the cross-linked chitosan with positive charges and heterotrophic bacteria using organic carbon sources as the double microcarriers, which not only can eliminate the adverse effect of the cross-linked chitosan on nitrifying bacteria, but also can enable the nitrifying bacteria to be adsorbed on the double microcarriers for rapid growth, thereby improving the culture density of the nitrifying bacteria.
(2) When the heterotrophic bacteria in the double microcarriers provided by the invention are used for culturing nitrifying bacteria, the biological activity of the nitrifying bacteria can gradually lose the final cell death under the conditions of no organic carbon source and aeration, the nitrifying bacteria can gradually replace the binding site of the original heterotrophic bacteria, and the nitrifying bacteria can be adsorbed on the carriers for rapid adaptive growth, so that the culture density can be improved.
Detailed Description
The process and effects of the present invention are described in further detail by the following examples. The embodiments and specific operation procedures are given on the premise of the technical scheme of the invention, but the protection scope of the invention is not limited to the following embodiments.
The experimental methods in the following examples, unless otherwise specified, are all conventional in the art. The experimental materials used in the examples described below were purchased from biochemical reagent stores unless otherwise specified.
The nitrifying bacteria culture density of the invention refers to the number of nitrifying bacteria per unit volume.
Example 1
The preparation method of the calcium carbonate-embedded chitosan microcarrier comprises the following specific steps: firstly, 2% (W/V) chitosan is dissolved in 1% (V/V) acetic acid solution to add CaCO 500mL 3 10g of nano particles, adding edible oil with the volume of 5 times, and adding 10mL of span-80 for vigorous stirring. Genipin was added to a final concentration of 20mM in the aqueous phase, stirring was continued for 24 hours, the precipitate was centrifuged, and washed with acetone, hot water, cold water several times to remove the oil phase and impurities remaining on the microsphere surface. Finally, acetone is used for dehydration for 2 times, and the obtained product is dried at room temperature to obtain yellowish powder, namely the chitosan microcarrier embedded with calcium carbonate.
And (3) adding the chitosan microcarrier with the calcium carbonate embedded in the shell into a culture system of candida tropicalis utilizing xylose for adsorption growth, wherein the mass concentration of the added xylose is 2mg/L. The culture conditions are as follows: the temperature is 25 ℃, the pH is 6.0-7.0, the shaking table fermentation culture is carried out, and the shaking table culture rotating speed is 200r/min. Culturing for 48h, taking out the solid, and drying at 40deg.C for 3h to obtain double microcarrier. The heterotrophic bacteria account for 30% of the carrier through detection.
Preparing 100mL of nitrifying bacteria enrichment culture solution in a laboratory, and subpackaging the nitrifying bacteria enrichment culture solution into 2 shake flasks of 200mL, wherein one shake flask is added with the carrier according to the ratio of 3g/L of the nitrifying bacteria enrichment culture solution to the culture solution in a nitrifying bacteria culture system, and one shake flask is a control group without the carrier; then inoculating the activated nitrifying bacteria into two shake flasks according to the volume ratio of 5% of the nitrifying bacteria to the culture solution in the nitrifying bacteria enrichment culture system, and carrying out enrichment culture on nitrifying bacteria by adopting a batch water exchange and drainage mode. The enrichment culture conditions of nitrifying bacteria are as follows: the temperature is 32 ℃, the pH is 7.8, the shake culture is carried out by a shaking table, and the concentration of dissolved oxygen is controlled to be 2mg/L. After 30 days of culture, the number of microorganisms was analyzed, and the culture density was 5 times that of the culture using no double microcarriers.
Example 2
The preparation method of the calcium carbonate-embedded chitosan microcarrier comprises the following specific steps: firstly, dissolving chitosan with the weight-volume ratio of 2% in acetic acid solution with the volume ratio of 1% for 500mL, adding CaCO 3 10g of nano particles, adding edible oil with the volume of 5 times, and adding 10mL of span-80 for vigorous stirring. 10mL of 25% glutaraldehyde was added, stirring was continued for 2 hours, 5g of sodium borohydride was added to react for 2 hours, and the precipitate was centrifugally separated and washed with acetone, hot water, and cold water several times to remove the oil phase and impurities remaining on the microsphere surface. Finally, acetone is used for dehydration for 2 times, and the obtained product is dried at room temperature to obtain yellowish powder which is the chitosan microcarrier embedded with calcium carbonate.
And (3) adding the calcium carbonate-embedded chitosan microcarrier into a culture system of candida tropicalis utilizing xylose for adsorption growth, wherein the mass concentration of the added xylose is 3g/L. The culture conditions are as follows: the temperature is 25 ℃, the pH is 6.0-7.0, the shaking table fermentation culture is carried out, and the shaking table culture rotating speed is 200r/min. Culturing for 48h, taking out the solid, and drying at 40deg.C for 3h to obtain double microcarrier. The heterotrophic bacteria account for 26% of the carrier after detection.
Preparing 100mL of nitrifying bacteria enrichment culture solution in a laboratory, and subpackaging the nitrifying bacteria enrichment culture solution into 2 shake flasks of 200mL, wherein one shake flask is added with the carrier according to the ratio of 3g/L of the nitrifying bacteria enrichment culture solution to the culture solution in a nitrifying bacteria culture system, and one shake flask is a control group without the carrier; then inoculating the activated nitrifying bacteria into two shake flasks according to the volume ratio of 5% of the nitrifying bacteria to the culture solution in the nitrifying bacteria enrichment culture system, and carrying out enrichment culture on nitrifying bacteria by adopting a batch water exchange and drainage mode. The enrichment culture conditions of nitrifying bacteria are as follows: the temperature is 32 ℃, the pH is 7.8, the shake culture is carried out by a shaking table, and the concentration of dissolved oxygen is controlled to be 2mg/L. After 30 days of culture, the number of microorganisms was analyzed, and the culture density was 4.5 times that of the culture using no double microcarriers.
Example 3
The difference from example 1 is that: the organic carbon source adopts glucose, and the heterotrophic bacteria are lactobacillus. The heterotrophic bacteria account for 10% of the carrier through detection. The carrier was used for enrichment culture of nitrifying bacteria for 30 days, and the culture density was 4.7 times that of the unused double microcarriers.
Example 4
The difference from example 1 is that: the organic carbon source adopts sucrose, and the heterotrophic bacteria are desulfurization monad. The heterotrophic bacteria account for 20% of the carrier through detection. The carrier was used for enrichment culture of nitrifying bacteria for 30 days, and the culture density was 4.3 times that of the unused double microcarriers.
Example 5
The difference from example 1 is that: the culture conditions are stationary culture, and the stationary culture is stirred every 60 min. The heterotrophic bacteria account for 25% of the carrier through detection. The carrier was used for enrichment culture of nitrifying bacteria for 30 days, and the culture density was 4.8 times that of the unused double microcarriers.
Comparative example 1
The difference from example 1 is that: directly adopting the prepared cross-linked chitosan for enrichment culture. After 30 days of enrichment culture of the vector for nitrifying bacteria, the number of microorganisms was analyzed, and the culture density using the double microcarriers was 1.4 times that of the culture density without using the double microcarriers.
Comparative example 2
The difference from example 1 is that: heterotrophic denitrifying bacteria are used to replace yeast. The heterotrophic bacteria account for 15% of the carrier through detection. After 30 days of enrichment culture of the vector for nitrifying bacteria, the number of microorganisms was analyzed, and the culture density using the double microcarriers was 1.5 times that of the culture density without using the double microcarriers.
Claims (20)
1. The preparation method of the double microcarriers for culturing nitrifying bacteria is characterized by comprising the following steps: preparing a cross-linked chitosan carrier embedded with calcium carbonate; and (3) adding the cross-linked chitosan carrier into a heterotrophic bacteria culture system utilizing an organic carbon source for adsorption growth, culturing until the late growth stage is stopped, taking out a solid and drying to obtain the double microcarriers.
2. The method according to claim 1, characterized in that: the calcium carbonate-embedded crosslinked chitosan carrier adopts a direct crosslinking or a chemical modification mode in crosslinking, and a crosslinking agent used for the direct crosslinking is at least one of epichlorohydrin, glutaraldehyde, formaldehyde, crown ethers and genipin.
3. The method according to claim 2, characterized in that: the cross-linking agent used for direct cross-linking is genipin.
4. The method according to claim 1, characterized in that: the heterotrophic bacteria are at least one of saccharomycetes, lactobacillus and sulfate reducing bacteria which utilize organic carbon sources.
5. The method according to claim 4, wherein: the heterotrophic bacteria are yeasts.
6. The method according to claim 4 or 5, characterized in that: the microzyme is at least one of candida, cryptococcus, hansenula, pichia, rhodotorula, torulopsis or candida; the lactobacillus is at least one selected from lactobacillus, bifidobacterium and lactococcus; the sulfate reducing bacteria are at least one selected from the group consisting of desulfurization unit cell bacteria and desulfurization line bacteria.
7. The method according to claim 6, wherein: the yeast is selected from candida tropicalis.
8. The method according to claim 1, characterized in that: the organic carbon source is at least one of glucose, xylose, sucrose and starch.
9. The method according to claim 1 or 8, characterized in that: the organic carbon source is added according to the mass concentration of 1-5g/L in the system after the addition.
10. The method according to claim 1 or 4, characterized in that: the culture conditions of the heterotrophic bacteria are as follows: the temperature is 20-38 ℃, and the pH is 6.0-8.5; and (3) carrying out stationary fermentation or shake culture, wherein stirring is carried out every 30-60min, and the rotation speed of the shake culture is 200-600r/min.
11. The method according to claim 10, wherein: the culture conditions of the heterotrophic bacteria are as follows: the temperature is 20-30deg.C, and the pH is 6.0-7.0.
12. The method according to claim 1, characterized in that: culturing for 24-80 h until the late logarithmic growth phase.
13. The method according to claim 1, characterized in that: the drying temperature is 25-50 ℃ and the drying time is 1-5h.
14. A dual microcarrier for culturing nitrifying bacteria, characterized in that: is prepared by the method of any one of claims 1-13.
15. The dual microcarrier of claim 14, wherein: the double microcarrier takes crosslinked chitosan as a matrix, and heterotrophic bacteria are adsorbed and grown on the matrix, wherein the heterotrophic bacteria account for 5-50% of the carrier.
16. The dual microcarrier of claim 15, wherein: heterotrophic bacteria account for 10% -30% of the carrier.
17. A culture method for improving the culture density of nitrifying bacteria is characterized by comprising the following steps: is prepared by the method of any one of claims 1-13 or 14-16, and is added into a nitrifying bacteria enrichment culture system for culturing nitrifying bacteria.
18. The method according to claim 17, wherein: the ratio of the adding amount of the double microcarriers to the culture solution in the nitrifying bacteria enrichment culture system is 1-5g/L.
19. The method according to claim 17, wherein: the volume ratio of the adding amount of the nitrifying bacteria to the culture solution in the nitrifying bacteria enrichment culture system is 0.5-5%.
20. The method according to claim 17, wherein: the nitrifying bacteria enrichment culture adopts batch water changing and draining or batch material supplementing; the enrichment culture conditions of nitrifying bacteria are as follows: the temperature is 28-35 ℃, the pH is 7.2-8.2, and the dissolved oxygen is 1-5mg/L.
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