CN110002609B - Microbial water quality improver and preparation method thereof - Google Patents
Microbial water quality improver and preparation method thereof Download PDFInfo
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- CN110002609B CN110002609B CN201910313772.3A CN201910313772A CN110002609B CN 110002609 B CN110002609 B CN 110002609B CN 201910313772 A CN201910313772 A CN 201910313772A CN 110002609 B CN110002609 B CN 110002609B
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 117
- 230000000813 microbial effect Effects 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 56
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 37
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 37
- 239000007788 liquid Substances 0.000 claims abstract description 37
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- 239000004005 microsphere Substances 0.000 claims abstract description 32
- 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 abstract description 30
- 239000008103 glucose Substances 0.000 claims abstract description 30
- 239000000725 suspension Substances 0.000 claims abstract description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000003607 modifier Substances 0.000 claims abstract description 23
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 19
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 19
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 14
- 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 claims abstract description 11
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 11
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 11
- 239000000661 sodium alginate Substances 0.000 claims abstract description 11
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 11
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 34
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 30
- 238000002156 mixing Methods 0.000 claims description 24
- 239000001110 calcium chloride Substances 0.000 claims description 17
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 17
- 239000000017 hydrogel Substances 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 15
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 230000004913 activation Effects 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 235000015278 beef Nutrition 0.000 claims description 15
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 15
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 235000010469 Glycine max Nutrition 0.000 claims description 12
- 244000068988 Glycine max Species 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 12
- 238000009630 liquid culture Methods 0.000 claims description 12
- 239000012137 tryptone Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 241000194108 Bacillus licheniformis Species 0.000 claims description 9
- 241000194103 Bacillus pumilus Species 0.000 claims description 9
- 244000063299 Bacillus subtilis Species 0.000 claims description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 9
- 239000011248 coating agent Substances 0.000 claims description 9
- 238000000576 coating method Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 238000010790 dilution Methods 0.000 claims description 9
- 241000194107 Bacillus megaterium Species 0.000 claims description 8
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 8
- 239000000084 colloidal system Substances 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000004132 cross linking Methods 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 8
- 238000003760 magnetic stirring Methods 0.000 claims description 8
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims 2
- 230000001580 bacterial effect Effects 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 9
- 230000006872 improvement Effects 0.000 abstract description 8
- 230000004083 survival effect Effects 0.000 abstract description 5
- 239000000969 carrier Substances 0.000 abstract description 3
- 230000015556 catabolic process Effects 0.000 abstract description 3
- 238000006731 degradation reaction Methods 0.000 abstract description 3
- 235000019422 polyvinyl alcohol Nutrition 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract 1
- 238000012807 shake-flask culturing Methods 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 16
- 238000009360 aquaculture Methods 0.000 description 13
- 244000144974 aquaculture Species 0.000 description 13
- 238000001914 filtration Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000004129 EU approved improving agent Substances 0.000 description 2
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- 238000002474 experimental method Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 238000001878 scanning electron micrograph Methods 0.000 description 1
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Classifications
-
- 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
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
-
- 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
- C02F2003/001—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms
- C02F2003/003—Biological treatment of water, waste water, or sewage using granular carriers or supports for the microorganisms using activated carbon or the like
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2305/00—Use of specific compounds during water treatment
- C02F2305/06—Nutrients for stimulating the growth of microorganisms
Abstract
The invention discloses a microbial water quality modifier and a preparation method thereof, wherein the preparation method comprises the following steps: (1) preparing carbon microspheres; (2) preparing a plate culture medium; (3) inoculating; (4) performing plate culture; (5) performing shake flask culture; (6) preparing a suspension liquid; (7) preparing a spherule microorganism water quality modifier. The invention adopts glucose, polyxylose and mannose as mixed carbon sources to prepare high-quality carbon microspheres as carriers of the strains, the strains are reasonably fixed, the survival rate is high, and stable bacterial colonies are formed. The bacterial colonies are fixed by using polyvinyl alcohol, sodium alginate and silicon dioxide, the degradation efficiency of bacillus to COD is ensured, and the resistance of the bacillus bacterial colonies to toxic and harmful substances is improved, so that the water quality improvement effect of the culture water area is improved.
Description
Technical Field
The invention relates to the technical field of water quality improvement, in particular to a microbial water quality improver and a preparation method thereof.
Background
With the rapid development of the aquaculture industry, the malignant influence of unreasonable cultivation behaviors and the increasingly expanded cultivation scale on the water quality is more and more serious. The good ecological environment of the aquaculture water areas is a necessary condition for survival and reproduction of aquatic products, and in the artificial aquaculture process, when the water quality conditions of the aquaculture water areas such as ponds and the like are poor, the normal growth and development and reproduction quality of the aquatic products can be influenced, even the death of the aquatic products can be caused, great economic loss can be caused, and the physical health of human can also be indirectly influenced. Solving the problem of water quality of artificial aquaculture water body has become a necessary requirement for developing green, healthy, harmless and efficient ecological aquaculture industry.
The function of the microbial water quality modifier in aquaculture is different from that of drugs, the microbial water quality modifier can evolve aquaculture water to achieve sustainable reuse of water resources, full absorption and conversion of feed are enhanced, water algae balance is improved through adjustment of the microbial water quality modifier on water quality, safe and pollution-free healthy edible aquatic products are obtained, and ecological balance is protected.
In the prior art, the product of the microbial water quality modifier is usually liquid or solid powder, and the microbial water quality modifier is directly put into water when in use. The loss of the microbial strains is serious, the survival rate is low, and a stable colony structure is difficult to form, so that the water quality improvement effect is poor, and the quality of the aquaculture water is difficult to improve.
Disclosure of Invention
The invention provides a microbial water quality improver and a preparation method thereof, and aims to solve the problem that a microbial strain in the microbial water quality improver is difficult to survive and runs off seriously, so that the water quality improvement effect is poor.
The preparation method of the microbial water quality modifier provided by the invention comprises the following steps:
(1) adding deionized water into glucose, polyxylose and mannose for dissolving, adding ferric trichloride for mixing, transferring into a reaction kettle, carrying out hydrothermal reaction for 10-15h at the temperature of 150-;
(2) preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and respectively pouring the plates after sterilization;
(3) inoculating bacillus on a flat plate by using an aseptic technique, uniformly coating, and selecting three appropriate dilutions to inoculate the bacillus on a beef extract peptone agar culture medium to obtain a bacillus flat plate;
(4) culturing the bacillus plate at a constant temperature of 37 ℃ for 1-2d to obtain bacillus strains;
(5) preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step (1) into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step (4), and culturing the bacillus strain in a shaking flask at the constant temperature of 37 ℃ for 1-2d at the rpm of 130-;
(6) centrifuging the bacillus strain turbid liquid obtained in the step (5) for 10-15min, and then discarding the supernatant to obtain a suspension liquid;
(7) mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, uniformly stirring until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, uniformly mixing, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, continuously stirring into small balls by magnetic stirring, crosslinking for 18-24h at the temperature of 4-6 ℃, and then washing the small balls for 2-3 times by using distilled water to obtain the microbial water quality modifier.
Optionally, step (7) further comprises activation, said step (7):
mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, stirring uniformly until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, mixing uniformly, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, stirring continuously by magnetic stirring to form small balls, crosslinking for 18-24h at the temperature of 4-6 ℃, washing the small balls for 2-3 times by using distilled water, transferring the small balls into a culture water body for activation culture, adopting an intermittent culture mode, taking 24h as an activation period, replacing a fresh culture water body after the period is over, and obtaining the microbial water quality modifier until the removal rate of COD reaches a high stable value.
Optionally, the bacillus comprises bacillus subtilis, bacillus megaterium, bacillus pumilus and bacillus licheniformis.
Optionally, in the step (1), NaCl and MgCl are further added into the reaction kettle2Or/and CaCl2。
Optionally, in step (1), the molar ratio of glucose, polyxylose and mannose is: 1: (0.5-1): (0.5-1).
Optionally, in the step (1), the concentration of the glucose is 0.2-0.8 mol/L.
The invention also provides the microbial water quality modifier prepared by the preparation method.
The invention has the following beneficial effects:
on one hand, the invention adopts glucose, polyxylose and mannose as a mixed carbon source to prepare the monodisperse carbon microsphere with the particle size of 5-10 mu m under the condition of not adding a template agent and a surfactant. The carbon microspheres are used as carriers of the strains, the strains are reasonably fixed, the survival rate is high, and stable bacterial colonies are formed; on the other hand, the bacterial colony is fixed by using polyvinyl alcohol, sodium alginate and silicon dioxide, the degradation efficiency of bacillus to COD is ensured, and the resistance of the bacillus bacterial colony to toxic and harmful substances is improved, so that the water quality improvement effect of the culture water area is improved.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Detailed Description
The invention provides a microbial water quality improver and a preparation method thereof, and aims to solve the problem that a microbial strain in the microbial water quality improver is difficult to survive and runs off seriously, so that the water quality improvement effect is poor. The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
The preparation method of the microbial water quality modifier provided by the invention comprises the following steps:
step S001: taking glucose, polyxylose and mannose with a molar ratio of 1:0.5:0.5, adding deionized water to dissolve the glucose, the polyxylose and the mannose to ensure that the concentration of the glucose is 0.5mol/L, and adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. Transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12h in a vacuum drying oven at 180 ℃, washing, filtering, and then carrying out 80 DEG CDrying for 12h to obtain the carbon microsphere.
Step S002: preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and pouring the plates after sterilization.
Step S003: inoculating the bacillus on a plate by using an aseptic technique, uniformly coating, and selecting three appropriate dilutions to inoculate the bacillus on a beef extract peptone agar culture medium to obtain the bacillus plate.
Step S004: culturing the bacillus plate at the constant temperature of 37 ℃ for 1-2d to obtain bacillus strains.
Step S005: preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step S001 into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step S004, and culturing the bacillus strain in a shake flask at the constant temperature of 37 ℃ for 1-2d at the rpm of 130-.
Step S006: and (4) centrifuging the bacillus strain turbid liquid obtained in the step (S005) for 10-15min, and then discarding the supernatant to obtain a suspension liquid.
Step S007: mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, uniformly stirring until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, uniformly mixing, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, continuously stirring into small balls by magnetic stirring, crosslinking for 18-24h at the temperature of 4-6 ℃, and then washing the small balls for 2-3 times by using distilled water to obtain the microbial water quality modifier.
The steps S001 to S007 described in the above embodiments do not limit the sequence of the steps, wherein the process flow may be changed according to the requirements in the specific implementation process, and are not described in detail herein.
To increase the activity of the strain, step S007 further comprises activation of the strain, alternatively: mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, stirring uniformly until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, mixing uniformly, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, stirring continuously by magnetic stirring to form small balls, crosslinking for 18-24h at the temperature of 4-6 ℃, washing the small balls for 2-3 times by using distilled water, transferring the small balls into a culture water body for activation culture, adopting an intermittent culture mode, taking 24h as an activation period, replacing a fresh culture water body after the period is over, and obtaining the microbial water quality modifier until the removal rate of COD reaches a high stable value.
The following examples can be referred to for specific embodiments of the preparation of the microbial water quality modifier: example 1
The preparation method of the microbial water quality improver provided in the embodiment 1 comprises the following steps:
step S101: taking glucose, polyxylose and mannose with a molar ratio of 1:0.5:0.5, adding deionized water to dissolve the glucose, the polyxylose and the mannose to ensure that the concentration of the glucose is 0.5mol/L, and adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. And transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12 hours in a vacuum drying oven at 180 ℃, washing, filtering, and drying for 12 hours at 80 ℃ to obtain the carbon microspheres.
Step S102: preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and pouring the plates after sterilization.
Step S103: inoculating bacillus subtilis, bacillus megaterium, bacillus pumilus and bacillus licheniformis on a plate by an aseptic operation technology, uniformly coating, and inoculating the bacillus subtilis, the bacillus megaterium, the bacillus pumilus and the bacillus licheniformis on a beef extract peptone agar culture medium by selecting three proper dilutions to obtain a bacillus plate.
Step S104: culturing the bacillus plate at the constant temperature of 37 ℃ for 2d to obtain bacillus strains.
Step S105: preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step S101 into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step S104, and culturing the bacillus strain at the constant temperature of 37 ℃ for 2d in a shaking flask at 150rpm to obtain a bacillus strain turbid solution.
Step S106: centrifuging the bacillus strain turbid liquid obtained in the step S105 for 10min, and then discarding the supernatant to obtain a suspension liquid, wherein the total bacteria amount in each milliliter of suspension liquid is 5 multiplied by 108～7×108And (4) respectively.
Step S107: mixing polyvinyl alcohol and water into a colloid in a water bath at 100 ℃, adding sodium alginate and silicon dioxide, uniformly stirring until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, uniformly mixing, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, continuously stirring into small balls by magnetic stirring, crosslinking at 5 ℃ for 20 hours, washing the small balls for 3 times by using distilled water, transferring the small balls into a culture water body for activation culture, adopting an intermittent culture mode, taking 24 hours as an activation period, and replacing with a fresh culture water body after the period is over until the removal rate of COD reaches a high stable value to obtain the microbial water quality modifier.
Example 2
The preparation method of another microbial water quality improver provided in this embodiment 2 includes the following steps:
step S201: taking glucose, polyxylose and mannose with a molar ratio of 1:0.5:0.5, adding deionized water to dissolve the glucose, the polyxylose and the mannose to ensure that the concentration of the glucose is 0.5mol/L, and adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. And transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12 hours in a vacuum drying oven at 180 ℃, washing, filtering, and drying for 12 hours at 80 ℃ to obtain the carbon microspheres.
Step S202: preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and pouring the plates after sterilization.
Step S203: inoculating bacillus subtilis on a plate by a sterile operation technology, uniformly coating, and selecting three appropriate dilutions to inoculate the bacillus subtilis on a beef extract peptone agar culture medium to obtain a bacillus plate.
Step S204: culturing the bacillus plate at the constant temperature of 37 ℃ for 2d to obtain bacillus strains.
Step S205: preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step S201 into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step S204, and culturing the bacillus strain at the constant temperature of 37 ℃ for 2d in a shaking flask at 150rpm to obtain a bacillus strain turbid solution.
Step S206: centrifuging the bacillus strain turbid solution obtained in the step S205 for 10min, and then discarding the supernatant to obtain a suspension liquid, wherein the total bacteria amount in each milliliter of suspension liquid is 5 multiplied by 108～7×108And (4) respectively.
Step S207: mixing polyvinyl alcohol and water into a colloid in a water bath at 100 ℃, adding sodium alginate and silicon dioxide, uniformly stirring until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, uniformly mixing, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, continuously stirring into small balls by magnetic stirring, crosslinking at 5 ℃ for 20 hours, washing the small balls for 3 times by using distilled water, transferring the small balls into a culture water body for activation culture, adopting an intermittent culture mode, taking 24 hours as an activation period, and replacing with a fresh culture water body after the period is over until the removal rate of COD reaches a high stable value to obtain the microbial water quality modifier.
Examples 3 to 5
Examples 3 to 5 provide a microbial water quality improver prepared in a similar manner to example 2 except that:
step S303 of embodiment 3: inoculating bacillus megatherium on a plate by an aseptic technique, uniformly coating, and selecting three appropriate dilutions to inoculate in a beef extract peptone agar medium to obtain a bacillus plate.
Step S403 of embodiment 4: inoculating the bacillus pumilus on a plate by an aseptic technique, uniformly coating, and inoculating the bacillus pumilus on a beef extract peptone agar culture medium by selecting three suitable dilutions to obtain a bacillus plate.
Step S503 of embodiment 5: inoculating bacillus licheniformis on the plate by an aseptic technique, uniformly coating, and selecting three appropriate dilutions to inoculate the bacillus licheniformis on a beef extract peptone agar medium to obtain a bacillus plate.
Comparative examples 1 to 2
The microbial water quality improver provided in comparative examples 1 to 2 was prepared similarly to example 1, except that:
step S101' of comparative example 1: taking glucose, adding deionized water for dissolution to ensure that the concentration of the glucose is 0.5mol/L, and then adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. And transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12 hours in a vacuum drying oven at 180 ℃, washing, filtering, and drying for 12 hours at 80 ℃ to obtain the carbon microspheres.
Step S201' of comparative example 2: taking glucose and polyxylose, adding deionized water for dissolving to ensure that the concentration of the glucose is 0.5mol/L, and then adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. And transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12 hours in a vacuum drying oven at 180 ℃, washing, filtering, and drying for 12 hours at 80 ℃ to obtain the carbon microspheres.
Comparative example 3
The preparation method of the microbial water quality improver provided by the comparative example 3 comprises the following steps:
step S301': taking glucose, polyxylose and mannose with a molar ratio of 1:0.5:0.5, adding deionized water to dissolve the glucose, the polyxylose and the mannose to ensure that the concentration of the glucose is 0.5mol/L, and adding FeCl3、NaCl、MgCl2、CaCl2Mixing glucose with FeCl3、NaCl、MgCl2、CaCl2In a molar ratio of 1:0.1:0.1:0.1: 0.1. And transferring the mixed solution into a reaction kettle, carrying out hydrothermal reaction for 12 hours in a vacuum drying oven at 180 ℃, washing, filtering, and drying for 12 hours at 80 ℃ to obtain the carbon microspheres.
Step S302': preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and pouring the plates after sterilization.
Step S303': inoculating bacillus subtilis, bacillus megaterium, bacillus pumilus and bacillus licheniformis on a plate by an aseptic operation technology, uniformly coating, and inoculating the bacillus subtilis, the bacillus megaterium, the bacillus pumilus and the bacillus licheniformis on a beef extract peptone agar culture medium by selecting three proper dilutions to obtain a bacillus plate.
Step S304': culturing the bacillus plate at the constant temperature of 37 ℃ for 2d to obtain bacillus strains.
Step S305': preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step S301 'into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step S304', and culturing the bacillus strain in a shake flask at the constant temperature of 37 ℃ for 2d at 150rpm to obtain a bacillus strain turbid solution.
Step S306': centrifuging the bacillus strain turbid liquid obtained in the step S305' for 10min, and then discarding the supernatant to obtain a suspension liquid, wherein the total bacteria amount in each milliliter of suspension liquid is 5 multiplied by 108～7×108And (4) respectively.
Step S307': and transferring the suspended bacterium liquid to an aquaculture water body for activation culture, adopting an intermittent culture mode, taking 24 hours as an activation period, and then changing the suspended bacterium liquid into a fresh aquaculture water body again after the period is finished until the COD removal rate reaches a high stable value to obtain the microbial water quality modifier.
From the scanning electron micrographs of the carbon microspheres prepared in comparative example 1 and comparative examples 1 to 2, it was found that the carbon microspheres obtained in example 1 were not only regular in shape and uniform in dispersion, but also the number of carbon microspheres having a particle size of 5 to 10 μm was the largest. The carbon microspheres in comparative example 1 are not regular in shape, and have both spherical, elliptical and dumbbell shapes, which indicates that the carbon microspheres prepared from a single carbon source are more sticky and have poor dispersibility. The shapes of the carbon microspheres obtained in comparative example 1 were varied, so that the particle diameters were difficult to estimate, but it was confirmed that the size was larger than that of the carbon microspheres obtained in example 1. The carbon microspheres obtained in comparative example 2 were not regular and round in shape, and were mostly polyhedral, but the dispersibility was not much different from that of example 1, and the number of particles having a particle diameter of 15 to 20 μm was the largest. By aligning the above three kinds of carbon microspheresThe specific surface area of the carbon microspheres obtained in example 1 was found to be the largest, indicating the highest adsorption capacity. Thus, the combination of three carbon sources, plus FeCl3、NaCl、MgCl2、CaCl2The obtained carbon microspheres are most suitable for being used as carriers of strains.
In addition, NaCl and MgCl are not added2、CaCl2Experiments with two or one of them have found that the prepared carbon microspheres have relatively poor dispersibility, and thus, NaCl and MgCl2、CaCl2The whole carbon microspheres are added to prepare the carbon microspheres, which is the preferred scheme provided by the invention.
In order to prove the function of the microbial water quality modifier in the aspect of aquaculture. And 8 identical four fish culture ponds are selected for testing. Wherein, No. 1-5 culture ponds are test ponds, and the water quality improving agents provided by examples 1-5 are correspondingly applied, No. 6-8 culture ponds are comparison ponds, and the water quality improving agents provided by comparative examples 1-3 are correspondingly applied. The application standard is that 0.5L of the suspension liquid is applied per mu per time (examples 1-5 correspond to pellets prepared by using 0.5L of the suspension liquid) calculated by using the suspension liquid obtained in the corresponding step 6. Administered once every half month.
The experiments show that the COD of the aquaculture water in the ponds No. 1-8 is reduced, but the reduction degree is different. Comparing example 1 with comparative examples 1-2, the COD reduction degree is from high to low, which is that example 1 is larger than comparative example 2 is larger than comparative example 1. The quality of the carbon microspheres influences the finally prepared microbial water quality modifier, and only when the carbon microspheres are regular in shape, small in size and high in dispersion degree, the specific surface area of the carbon microspheres is suitable for adsorption of strains, the strains are reasonably fixed, the survival rate is high, stable bacterial colonies are formed, and the water quality improvement effect of a culture water area is improved. The comparison of examples 1-5 shows that the water quality treatment effect of the mixed strains of bacillus subtilis, bacillus megaterium, bacillus pumilus and bacillus licheniformis is the best, and the mixed strains are preferably used for preparing the microbial water quality modifier. The lowest COD reduction degree in the No. 1-8 pool is the No. 8 pool, and accordingly, the comparison between the example 1 and the comparative example 3 shows that the bacterial colony is fixed by using polyvinyl alcohol, sodium alginate and silicon dioxide in the example 1, the degradation efficiency of bacillus to COD is ensured, the resistance of the bacterial colony of the bacillus to toxic and harmful substances is improved, and the water quality improvement effect of the culture water area is improved.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure herein. This invention is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (5)
1. The preparation method of the microbial water quality improver is characterized by comprising the following steps:
(1) dissolving glucose, polyxylose and mannose in deionized water, adding FeCl3、NaCl、MgCl2、CaCl2Mixing, transferring to a reaction kettle, carrying out hydrothermal reaction for 10-15h in a vacuum drying box at the temperature of 150-;
(2) preparing 100mL of beef extract peptone agar plate culture medium according to the formula of the culture medium, and respectively pouring the plates after sterilization;
(3) inoculating bacillus on a plate by using an aseptic technique, uniformly coating, and selecting three suitable dilutions to inoculate the bacillus on a beef extract peptone agar culture medium to obtain a bacillus plate, wherein the bacillus comprises bacillus subtilis, bacillus megaterium, bacillus pumilus and bacillus licheniformis;
(4) culturing the bacillus plate at a constant temperature of 37 ℃ for 1-2d to obtain bacillus strains;
(5) preparing a tryptone soybean broth liquid culture medium, adding the carbon microspheres obtained in the step (1) into the tryptone soybean broth liquid culture medium, inoculating the bacillus strain obtained in the step (4), and culturing the bacillus strain in a shaking flask at the constant temperature of 37 ℃ for 1-2d at the rpm of 130-;
(6) centrifuging the bacillus strain turbid liquid obtained in the step (5) for 10-15min, and then discarding the supernatant to obtain a suspension liquid;
(7) mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, uniformly stirring until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, uniformly mixing, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, continuously stirring into small balls by magnetic stirring, crosslinking for 18-24h at the temperature of 4-6 ℃, and then washing the small balls for 2-3 times by using distilled water to obtain the microbial water quality modifier.
2. The method of claim 1, wherein step (7) further comprises activation, and wherein step (7):
mixing polyvinyl alcohol and water into a colloid in a water bath at the temperature of 80-100 ℃, adding sodium alginate and silicon dioxide, stirring uniformly until all the polyvinyl alcohol and the water are dissolved to prepare hydrogel, cooling to room temperature for later use, adding a suspension liquid into the hydrogel, mixing uniformly, slowly dripping the suspension liquid into a saturated boric acid solution by using an injector, stirring continuously by magnetic stirring to form small balls, crosslinking for 18-24h at the temperature of 4-6 ℃, washing the small balls for 2-3 times by using distilled water, transferring the small balls into a culture water body for activation culture, adopting an intermittent culture mode, taking 24h as an activation period, replacing a fresh culture water body after the period is over, and obtaining the microbial water quality modifier until the removal rate of COD reaches a high stable value.
3. The method according to claim 1, wherein in the step (1), the molar ratio of glucose, polyxylose and mannose is: 1: (0.5-1): (0.5-1).
4. The production method according to claim 3, wherein in the step (1), the concentration of glucose is 0.2 to 0.8 mol/L.
5. A microbial water quality improver produced by the production method according to any one of claims 1 to 4.
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