CN114292762B - Candida palmi and application thereof - Google Patents
Candida palmi and application thereof Download PDFInfo
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Abstract
The invention relates to candida palmatium (Candida palmioleophila) which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.22737. The screened candida palmatine has heterotrophic nitrification and aerobic denitrification functions, can resist high-concentration nitrite and has excellent degradation effect, and the candida palmatine can reach higher viable count when grown on a culture medium with initial pH value of 4.0-8.0, and has wide acid-base tolerance and strong adaptability to severe environments.
Description
Technical Field
The invention relates to candida palmatium and application thereof, in particular to candida palmatium which is particularly suitable for degrading ammonia nitrogen, nitrite nitrogen and total nitrogen in cultivation wastewater, and belongs to the technical field of environmental microorganisms.
Background
Since the innovation was opened, the aquaculture industry in China rapidly developed under the guidance of science, and the aquaculture yield in the prior art exceeds the fishing yield, so that the aquaculture industry in China walks on an intensive and industrial aquaculture road for improving the aquaculture yield. Under the high-density cultivation mode, the water environment of cultivation deteriorates, and the self-cleaning capacity of the water body is exceeded, so that the sewage treatment demands are gradually increased. Research data shows that in the bait for breeding animals, the assimilation and utilization efficiency of nitrogen salts is only 30%, a large amount of organic nitrogen and inorganic nitrogen are remained in excrement and residual bait, and excessive bait cannot be completely absorbed and utilized by aquatic animals, so that a large amount of organic wastes are deposited, the self-purifying capacity of the water body is weakened, and the concentration of pollutants such as ammonia nitrogen, nitrite nitrogen and the like in the water body is increased.
According to the related report, when the nitrogen content in the water body is more than 0.2mg/L, the water body eutrophication condition is provided. In the nitrogen-containing compound, ammonia nitrogen and nitrite nitrogen have great influence on aquaculture organisms, the aquatic organisms are very sensitive to ammonia, and if the ammonia with too high concentration enters the aquaculture animals through gills and involucra, the gill epidermis cells are damaged, the oxygen carrying capacity of blood is reduced, the physiological and biochemical reactions of the aquatic organisms are influenced, and the aquatic animals die when serious suffocation occurs. Nitrite with a certain concentration has toxic action on aquatic animals, and is mainly reflected in the reaction with hemoglobin in blood of the aquatic animals, so that the oxygen transporting capacity of the aquatic animals is reduced, the tissue of the cultivated animals is anoxic and choked to death, and the growth and development of the aquatic animals are influenced, and meanwhile, the aquatic animals are seriously threatened to the life health of human beings. There is an article showing that nitrous acid reacts with secondary amines generated by human protein decomposition to form nitrosamines, which are carcinogens with teratogenicity and embryotoxicity, upon ingestion of sufficient amounts of nitrite by humans. Nitrite can cause intestinal-derived blue-violet disease, cause dyspnea, damage central nervous system, induce coma, convulsion, loss of consciousness and other symptoms.
At present, the denitrification technical method is mainly researched from three directions of physics, chemistry and biology. The biological denitrification technology is widely applied due to the advantages of good treatment effect, no secondary pollution, low treatment cost, convenient operation and management and the like, and becomes an effective denitrification method in water. Strains capable of degrading ammonia nitrogen and nitrite nitrogen efficiently exist in nature, and the strains with the characteristics are screened out manually, so that the growth characteristics, denitrification characteristics and application thereof in sewage denitrification are studied in depth, and the strain has important theoretical value and practical significance in improving sewage denitrification treatment efficiency and economical efficiency.
Chinese patent application CN202110333162.7 discloses heterotrophic nitrifying candida palmatium (Candida palmioleophila) capable of resisting high-salt environment, which is obtained by screening and separating from a heterotrophic nitrifying culture medium with a salt content of 9%, has a test effect in pharmaceutical and chemical wastewater with a salinity of 4600mg/L, has an ammonia nitrogen removal rate of 69.3% for 36 hours, has a total nitrogen removal rate of 52.9% for 42 hours, has a COD removal rate of 93.1% and is suitable for pharmaceutical and chemical wastewater with the characteristics of high salt and high concentration of organic pollutants, but has only heterotrophic nitrification, cannot perform aerobic denitrification at the same time, cannot degrade nitrous acid nitrogen in the cultivation wastewater with high nitrite nitrogen content, and is not suitable for purifying the cultivation wastewater with ammonia nitrogen, high nitrite nitrogen content and low salt.
Disclosure of Invention
Aiming at the current situation that the existing microbial strain is not suitable for cultivation wastewater containing ammonia nitrogen, high-content nitrite nitrogen and low salt, the invention provides candida palmatium suitable for cultivation wastewater/sewage purification, a microbial agent containing the candida palmatium and application thereof.
The invention claims and protects candida palmatium Candida palmioleophila NOB-1, wherein the 18S rDNA sequence is shown as SEQ ID No. 1, the 26S rDNA sequence is shown as SEQ ID No.2, and the candida palmatium is preserved in the China general microbiological culture collection center with the address: the preservation number of the Beijing city is CGMCC No.22737 and the preservation date is 2021, 6 and 21.
The invention also claims a microbial agent containing the candida palmatina.
The technical scheme of the invention has the beneficial effects that:
(1) The screened candida palmatii oil has heterotrophic nitrification and aerobic denitrification functions, and when the inoculum liquid amount is 1250ppm, the ammonia nitrogen degradation rate in the culture sewage reaches 99.3 percent after 48 hours, the nitrite degradation rate reaches 98.7 percent, and the total nitrogen degradation rate reaches 61.2 percent.
(2) The screened candida palmata can tolerate nitrite with high concentration and has excellent degradation effect, the degradation rate reaches more than 99% for nitrite nitrogen with the concentration of 220mg/L within 48 hours, and the nitrite nitrogen in the culture solution is negative after 72 hours and is qualitatively detected for nitrite nitrogen with the concentration of 300-400 mg/L.
(3) The screened candida palmatii oil can reach higher viable count when grown on a culture medium with the initial pH value of 4.0-8.0, and has wide acid-base tolerance and strong adaptability to severe environments.
(4) The screened candida palmatii oil has higher tolerance degree to salt content, and the viable count is not affected basically when the candida palmatii oil grows in a culture medium with NaCl content below 25 per mill.
The preparation method of the microbial agent comprises the following steps:
(1) Activating strains: inoculating candida palmatine Candida palmioleophila NOB-1 into nutrient broth culture medium, and culturing at 25-35 ℃ and 150-300rpm for 12-48 hours to obtain strain activation liquid;
(2) Fermentation: inoculating the strain activation solution obtained in the step (1) into the fermentation medium in the fermentation tank according to the inoculation amount of 5-15vol% after the fermentation medium in the fermentation tank is disinfected, controlling the temperature to be 25-35 ℃, fermenting under the conditions that the aeration ratio is (1-2): 1 and 150-300rpm, and stopping fermentation when dissolved oxygen starts to rise to obtain fermentation solution;
(3) Preparing a microbial agent: and (3) diluting the fermentation broth obtained in the step (2) to obtain the microbial agent.
Further, the composition of the fermentation medium is as follows: 15-30g/L of carbon source, 5-15g/L of nitrogen source and PO 4 3- 6-10g/L，K + 0.2-0.5g/L，Mg 2+ 0.03-0.1g/L，Na + 0.2-0.8g/L，Fe 2+ (5-15)*10 -3 g/L, the balance being water, and the pH value is 6.5-8;
preferably, the composition of the fermentation medium is as follows: 20-25g/L of carbon source, 8-12g/L of nitrogen source and PO 4 3- 7-9g/L，K + 0.2-0.4g/L，Mg 2+ 0.03-0.05g/L，Na + 0.3-0.5g/L，Fe 2+ (8-10)*10 -3 g/L, the balance being water, and the pH value is 6.5-7.5.
Further, the nutrient broth medium has the following composition: 10g/L peptone, 3g/L beef powder, 5g/L sodium chloride and the balance of water, and the pH is natural.
Further, the carbon source is selected from one or more of glucose, starch, sucrose, dextrin, sodium succinate and sodium citrate.
Further, the nitrogen source is selected from one or more of yeast extract powder, peptone, ammonium salt, urea and soybean meal powder, and the ammonium salt is preferably ammonium dihydrogen phosphate and ammonium sulfate.
Preferably, the K + The source of the catalyst is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride and potassium nitrate, and the Mg 2+ Is one or more of magnesium sulfate and magnesium chloride, and the Na is + The source of the Fe is one or more of sodium chloride, sodium sulfate, sodium nitrate, sodium carbonate, sodium acetate and sodium succinate 2+ The source of the catalyst is one or more of ferrous sulfate, ferrous chloride and ferrous ammonium sulfate.
The aeration ratio in the preparation method of the microbial agent refers to the ratio of the volume of air introduced into the fermentation tank to the total volume of fermentation liquid in each minute.
The invention also claims a method of purifying a water body using candida palmatii or a microbial agent comprising candida palmatii, comprising the step of inoculating candida palmatii or a microbial agent comprising candida palmatii into the water body.
Further, the pH of the water body is 4.0-8.0, the NaCl content of the water body is not more than 25 per mill, and preferably the NaCl content is not more than 15 per mill;
further, the concentration of nitrite nitrogen in the water body is not more than 400mg/L, preferably 220mg/L or less.
Further, the water body is culture sewage/wastewater.
The invention also claims the application of candida palmatium or a microbial agent containing candida palmatium in the field of water purification.
Further, the candida palmata or the microbial agent containing candida palmata is used for degrading nitrogen-containing substances in water, preferably substances containing ammonia nitrogen and nitrite nitrogen, more preferably substances containing nitrite nitrogen.
Detailed Description
The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.
Example 1 screening of strains and preliminary Performance evaluation
1.1 enrichment and screening of samples
Obtaining a sample from aerobic section activated sludge of a sewage treatment plant in Jiaonang area of Qingdao city, putting the sample into an Erlenmeyer flask containing 100ml of sterile water, adding glass beads, and vibrating until the sample is uniform; after shaking evenly, 10ml of supernatant is taken and added into sterilized primary enrichment medium for primary enrichment. After 72h, 10ml of primary enrichment liquid is sucked into a fresh secondary enrichment culture medium for secondary enrichment; and analogically obtaining a third-stage enrichment solution.
Three media were used in each stage of enrichment, each with 3 replicates, the composition of the enrichment medium was as follows:
culture medium (one): glucose 10g, ammonium sulfate 2.0g, sodium nitrite 0.75g, magnesium sulfate heptahydrate 1.0g, dipotassium phosphate dihydrate 1.0g, water 1000ml, ph=7.8.
Culture medium (two): 1g of ammonium sulfate, 3.4g of sodium succinate, 0.1g of magnesium sulfate heptahydrate, 1.5g of dipotassium hydrogen phosphate, 0.01g of manganese sulfate tetrahydrate, 0.01g of ferrous sulfate heptahydrate, 0.5g of sodium chloride, 0.5g of sodium nitrite, 1000mL of water and pH=7.8.
Culture medium (III): glucose 5.0g, sodium nitrite 2g, magnesium sulfate heptahydrate 0.5g, dipotassium hydrogen phosphate dihydrate 1.0g, sodium chloride 1.0g, water 1000ml, ph=7.8.
And (3) qualitatively detecting nitrite in the enrichment culture solution by using a Gris reagent.
The preparation method of the Gris reagent comprises the following steps:
grissii: dissolving 0.5g of sulfanilic acid in 50ml of 30% acetic acid under heating, preserving in the dark;
grissi II: 0.4g of 1-naphthol was boiled with 100ml of water, and 6ml of 80% acetic acid was added to the colorless solution poured out from the blue dreg.
The application method of the Gris reagent comprises the following steps: 200 mu L of sample to be detected is added into the centrifuge tube, 1 drop of Grignard I reagent is dripped into the centrifuge tube, 1 drop of Grignard II reagent is dripped into the centrifuge tube, and the centrifuge tube cover is covered. If the mixed solution shows pink color, the sample contains nitrite ions.
The experimental results are shown in Table 1, the negative control was deionized water, and the positive control was 1mg/L sodium nitrite solution.
TABLE 1 qualitative detection of nitrite Nitrogen content in enriched liquid at each level
As can be seen from table 1, parallel 1 and parallel 3 of the culture medium (three), and parallel 2 of the culture medium (two) show good adaptability and degradation capability to nitrite in the first, second and third enrichment processes, so that the third enrichment liquid of the above three groups is selected as a sample for separating and purifying functional strains.
1.2. Isolation and purification of functional strains
For the samples selected in the previous step, single colonies were tested and separated by using a gradient dilution and plate coating method, and then single colony purification was performed by using a multi-stage plate streaking method. By adopting the separation and purification method, 2 strains of single bacteria are finally obtained, which are respectively named NOB-1 and NOB-2, and are preserved in a refrigerator at 4 ℃ by adopting a bevel method.
1.3. Evaluation of Effect of functional Strain
After the two separated functional bacteria are activated for 24 hours by using a nutrient broth culture medium, inoculating the two functional bacteria into an evaluation culture medium containing nitrite nitrogen in an inoculum size of 0.2vol%, measuring the content of the nitrite nitrogen in the culture medium at regular time and calculating degradation rate, wherein the detection method of the nitrite nitrogen is implemented according to a spectrophotometry for measuring nitrite nitrogen in water quality of GB/T7493, and the results are shown in tables 2 and 3.
Evaluation of the medium formulation: glucose 5.0g, sodium nitrite 0.43g, magnesium sulfate heptahydrate 0.5g, dipotassium phosphate dihydrate 1.0g, sodium chloride 1.0g, water 1000ml, ph=7.8.
TABLE 2 evaluation of degradation of nitrite nitrogen in Medium by Strain NOB-1
TABLE 3 evaluation of degradation of nitrite nitrogen in Medium by Strain NOB-2
As can be seen from the table, the NOB-1 strain can degrade nitrite nitrogen to 0.24mg/L within 41h, the degradation rate reaches more than 99%, and the NOB-2 strain can degrade nitrite nitrogen to 2.1mg/L within 69h, and the degradation rate reaches more than 97%; the NOB-1 strain is subjected to glycerol tube freezing preservation at the temperature of minus 80 ℃ for further study.
Example 2 detection and identification of Strain NOB-1
2.1 Experimental methods
2.1.1 genomic DNA extraction
1) Taking a 1.5ml centrifuge tube, adding 200 mu L of pretreatment liquid and three glass beads, then adding a proper amount of bacteria sample, and putting into a grinder for grinding until the mixture is fully ground.
2) Adding 20 mu L of protease K solution, mixing uniformly, and standing at 37 ℃ for 30-60 min.
3) 200. Mu.L of lysate was added, mixed well by inversion and left at 70℃for 10min.
4) 200 mu L of absolute ethyl alcohol is added, fully inverted and uniformly mixed, and short centrifugation is carried out to remove liquid drops on the inner wall of the tube cover.
5) Passing through the adsorption column, washing with washing liquid for 1 time, and washing with rinsing liquid for 2 times.
6) The adsorption column is placed for 5 to 10 minutes at room temperature to thoroughly dry the residual rinse liquid in the adsorption material.
7) Transferring the adsorption into a new centrifuge tube, suspending and dropwise adding 50-100 mu L ddH2O to the middle position of the adsorption film, standing for 5-10 min at room temperature, centrifuging for 2min at 12,000rpm, and collecting the solution into the centrifuge tube.
2.1.2 16S/18S amplification
Table 4: primer information
Table 5: PCR amplification reaction System and conditions
2.2 PCR product detection and purification
The 3. Mu.L PCR products were subjected to 1.0% agarose gel detection, and the banding was observed.
The PCR product purification is operated according to the standard operation flow of magnetic bead purification, and the principle that the magnetic beads can adsorb or release charged substances is utilized to adsorb DNA in a high-salt low-pH solution and release DNA in a low-salt high-pH solution, so that the purpose of separating and purifying DNA products is achieved.
2.3 sequencing
And (3) performing on-machine detection on the purified PCR product, wherein the 18S rDNA sequence is shown as SEQ ID No. 1, the 26S rDNA sequence is shown as SEQ ID No.2, and comparing the PCR product with NCBI, and the identification result of the strain is candida palmatium Candida palmioleophila.
EXAMPLE 3 investigation of the tolerance of candida palmatina NOB-1 to environmental conditions
3.1 growth of the Strain NOB-1 at different pH values
Picking the one-ring fungus moss into a nutrient broth culture medium, and performing shake culture at 30 ℃ and 220rpm for 24 hours for activation; after activation, 10vol% of the inoculum was inoculated into each group of fresh sterilized fermentation media, and shake-cultured at 30℃for 48 hours at 150 rpm. The cultured fermentation broth was counted using a gradient dilution and plate coating method.
The formula of the fermentation medium comprises: glucose 20g, diammonium phosphate 10g, magnesium sulfate heptahydrate 0.5g, dipotassium phosphate dihydrate 1.0g, ferrous sulfate heptahydrate 0.04g, sodium chloride 1.0g, and water 1000mL, respectively adjusting pH=3, pH=4, pH=5, pH=6, pH=7, pH=8, and pH=9.
TABLE 6 growth of the strain NOB-1 at different pH values
As shown in Table 6, the strain NOB-1 can reach a higher viable count at an initial pH=4.0-8.0, has a wide acid-base tolerance and has a strong adaptability to severe environments.
3.2 evaluation of the tolerance of the Strain NOB-1 to different salinity
Picking the one-ring fungus moss into a nutrient broth culture medium, and carrying out shake culture at 30 ℃ and 220rpm for 24 hours for activation; after activation, the mixture was inoculated into each group of fresh sterilized fermentation media (sodium chloride content varies from 1 to 35%) at 5vol%, and the cultured fermentation broth was subjected to shaking culture at 35℃and 220rpm for 48 hours, and the culture broth was counted by using a gradient dilution and plate coating method.
The formula of the fermentation medium comprises: glucose 20g, diammonium phosphate 10g, magnesium sulfate heptahydrate 0.5g, dipotassium phosphate dihydrate 1.0g, ferrous sulfate heptahydrate 0.04g, sodium chloride 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 and 35.0g, water 1000mL, pH=6.5, respectively.
TABLE 7 tolerance of the strain NOB-1 to different salinity
| NaCl content | Viable count (CFU/ml) | NaCl content | Viable count (CFU/ml) |
| 1‰ | 7.9×10 8 | 20‰ | 6.9×10 8 |
| 5‰ | 7.8×10 8 | 25‰ | 5.5×10 8 |
| 10‰ | 7.9×10 8 | 30‰ | 0.8×10 8 |
| 15‰ | 7.2×10 8 | 35‰ | 0.2×10 8 |
As can be seen from the data in Table 7, the strain NOB-1 has a high tolerance to salt content, the decrease in viable count is not obvious under the condition of 25 permillage sodium chloride concentration, and the strain NOB-1 is sensitive to 30 permillage sodium chloride concentration or more, and thus, the tolerance to salinity of NOB-1 is about 25 permillage.
3.3 evaluation of degradation ability of Strain NOB-1 in high concentration nitrite Nitrogen
Picking the one-ring fungus moss into a nutrient broth culture medium, and carrying out shake culture at 30 ℃ and 220rpm for 24 hours for activation; preparing 30L of fermentation medium, and sequentially performing blank elimination and solid elimination in a 50L small test tank. After cooling, the activated liquid was inoculated into a small test tank at 15vol% of the inoculum size, and fermentation was performed at 30℃and 300rpm under the aeration ratio of (1-2): 1 for about 20 hours. Diluting with adjuvant to obtain viable bacteria with number of 3.0X10 8 CFU/mL bacterial liquid. Then adding the solution into an evaluation culture medium according to the inoculation amount of 0.2vol%, carrying out shaking culture at 30 ℃ and 220rpm, and then detecting the nitrite nitrogen content in the culture solution at fixed time, wherein the detection method of nitrite nitrogen is carried out according to the spectrophotometry for detecting nitrite nitrogen in water quality of GB/T7493, and the results are shown in Table 8.
The formulation of the fermentation medium is as follows: glucose 20g, diammonium phosphate 10g, magnesium sulfate heptahydrate 0.5g, dipotassium phosphate dihydrate 1.0g, ferrous sulfate heptahydrate 0.04g, sodium chloride 1.0g, water 1000ml, ph=6.0.
The formulation of the evaluation medium is as follows: glucose 5.0g, magnesium sulfate heptahydrate 0.5g, dipotassium hydrogen phosphate dihydrate 1.0g, sodium chloride 1.0g, sodium nitrite of experimental groups 1 to 5 were added in amounts of 0.25g, 0.5g, 1.0g, 1.5g and 2.0g, respectively, water 1000ml, ph=7.8.
TABLE 8 evaluation of degradation ability of high concentration nitrite nitrogen by Strain NOB-1
As shown in the data in Table 8, the degradation rate of the candida palmatii NOB-1 on nitrite nitrogen within 220mg/L reaches more than 99% in 48 hours, the candida palmatii NOB-1 has good degradation capability on nitrite nitrogen within 300-400mg/L, the degradation rate of the candida palmatii NOB-1 in 48 hours reaches 75.5% and 65.8% respectively, and the candida palmatii NOB-1 still has degradation capability after qualitative detection, and the nitrite nitrogen in the culture solution is negative after 72 hours.
Example 4 application of candida palmatii NOB-1 in aquaculture wastewater
4.1 preparation of candida palmata liquid
Picking the one-ring fungus moss into a nutrient broth culture medium, and performing shake culture for 24 hours at 30 ℃ and 220rpm for activation; then preparing 30L fermentation medium, and sequentially performing empty digestion and actual digestion in a 50L small test tank. Cooling, inoculating the activated liquid into small test tank according to 10vol% inoculum size, fermenting at 30deg.C and 300rpm under aeration ratio of (1-2): 1 for about 20 hr, discharging after fermentation, and diluting with appropriate amount of adjuvant to obtain 3.0X10 8 CFU/mL bacterial liquid.
4.2 treatment of the culture wastewater by the candida palmatii liquid
(1) Experiment site: a small laboratory of Ublue biological stock, ublue Innovative garden.
(2) The experimental procedure is as follows:
(1) 8L of cultivation sewage is taken and poured into an SBR reactor, a stirrer is started, the stirrer is started for 4 hours, the stirrer is closed for 4 hours, the intermittent operation is performed, an aeration device is started for 6 hours, the stirrer is closed for 2 hours, and the intermittent operation is performed.
(2) 10ml of bacterial liquid is added into the SBR reactor on the 1 st day, and 1000ml/d of fresh sewage is replaced by the SBR reactor every other day on the 3 rd day.
(3) The supernatant is taken every day, water quality indexes such as ammonia nitrogen, nitrite nitrogen, total nitrogen and the like are detected, the water quality change condition is monitored, the temperature of the sewage is controlled to be 25-35 ℃ in the whole experimental process, the ammonia nitrogen detection method is implemented according to the ' determination of HJ 535-2009 ammonia nitrogen-Nashi reagent spectrophotometry ', the detection methods of nitrite nitrogen and total nitrogen are respectively implemented according to the ' determination spectrophotometry of GB/T7493 water quality nitrite nitrogen ' and the ' determination of HJ 636 water quality total nitrogen ' alkaline potassium persulfate digestion ultraviolet spectrophotometry ', and the results are shown in Table 9.
TABLE 9 Water quality index variation of Candida palmis NOB-1 treated culture wastewater
As can be seen from the data in Table 9, the candida palmatii NOB-1 has certain degradation effects on ammonia nitrogen, nitrite nitrogen and total nitrogen in the culture sewage, the degradation rate of ammonia nitrogen reaches 99.3% at 48 hours, the degradation rate of nitrite reaches 98.7%, and the degradation rate of total nitrogen reaches 61.2%; the subsequent pollutant supplementation can reach more than 90% of degradation rate in 24 hours for ammonia nitrogen and nitrite nitrogen, and the degradation rate in 24 hours for total nitrogen can also reach 32.6% and 32.8% respectively.
Example 5 application of candida palmatium NOB-1 in chemical wastewater
5.1 preparation of candida palmata liquid
Picking the one-ring fungus moss into a nutrient broth culture medium, and performing shake culture for 24 hours at 30 ℃ and 220rpm for activation; then preparing 30L fermentation medium, and sequentially performing empty digestion and actual digestion in a 50L small test tank. Cooling, inoculating the activating solution into small test tank, fermenting at 25deg.C and 300rpm under aeration ratio of (1-2): 1 for about 20 hr, discharging after fermentation, adding appropriate amount of adjuvant, and diluting to obtain 3.0X10 8 CFU/mL bacterial liquid.
5.2 treatment of chemical wastewater by candida palmata liquid
(1) Experiment site: a small laboratory of Ublue biological stock, ublue Innovative garden.
(2) The experimental procedure is as follows:
(1) 8L of chemical wastewater is taken and poured into an SBR reactor, a stirrer is started, the stirrer is started for 4 hours, the stirrer is closed for 4 hours, the intermittent operation is performed, an aeration device is started for 6 hours, the stirrer is closed for 2 hours, and the intermittent operation is performed.
(2) 10ml of bacterial liquid is added into the SBR reactor on day 1, and 500ml/d of fresh wastewater is replaced by the SBR reactor every 2 days on day 4.
(3) The supernatant is filtered after being flocculated and precipitated by PAC and PAM, the temperature of the wastewater is controlled to be 25-35 ℃ in the whole experimental process, the ammonia nitrogen detection method is implemented according to the 'determination of HJ 535-2009 ammonia nitrogen-Nashi reagent spectrophotometry', the nitrite nitrogen and total nitrogen detection methods are respectively implemented according to the 'determination spectrophotometry of GB/T7493 water quality nitrite nitrogen' and the 'determination of HJ 636 water quality total nitrogen alkaline potassium persulfate digestion ultraviolet spectrophotometry', and the results are shown in Table 10.
5.3 Water quality Change Condition
TABLE 10 Water quality index variation of candida palmatina NOB-1 for treating chemical wastewater
As can be seen from the data in Table 10, the candida palmatii NOB-1 has certain degradation effects on ammonia nitrogen, nitrite nitrogen and total nitrogen in chemical wastewater, the degradation rate of ammonia nitrogen reaches 89.5% at 72h, the degradation rate of nitrite reaches 93.6%, and the degradation rate of total nitrogen reaches 64.9%; the subsequent pollutant supplementation can reach more than 85% of degradation rate of ammonia nitrogen in 48 hours, more than 96% of degradation rate of nitrite nitrogen in 48 hours, and 51.6% and 57.6% of degradation rate of total nitrogen in 48 hours respectively.
Therefore, the strain has good degradation effect on different forms of nitrogen pollutants in the water body, and has wide application scenes in sewage treatment.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
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ctgaggactg cgtctttgac taggatgctg gcataatgat cctataccgc ccgtc 595
Claims (7)
1. Candida palmatium (Candida palmioleophila) is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.22737.
2. The candida palmatina of claim 1 wherein the 18S rDNA sequence of candida palmatina is shown in SEQ ID No. 1 and the 26S rDNA sequence is shown in SEQ ID No. 2.
3. A microbial agent comprising the candida palmatina according to claim 1 or 2.
4. The method for producing a microbial agent according to claim 3, comprising the steps of:
(1) Activating strains: inoculating candida palmatii in nutrient broth culture medium, and culturing at 25-35 ℃ and 150-300rpm for 12-48 hours to obtain strain activation liquid;
(2) Fermentation: inoculating the strain activation solution obtained in the step (1) into the fermentation medium in the fermentation tank according to the inoculation amount of 5-15vol% after the fermentation medium in the fermentation tank is disinfected, controlling the temperature to be 25-35 ℃, fermenting under the conditions that the aeration ratio is (1-2): 1 and 150-300rpm, and stopping fermentation when dissolved oxygen starts to rise to obtain fermentation solution;
(3) Preparing a microbial agent: and (3) diluting the fermentation broth obtained in the step (2) to obtain the microbial agent.
5. The method according to claim 4, wherein the composition of the fermentation medium is as follows: 15-30g/L of carbon source, 5-15g/L of nitrogen source and PO 4 3- 6-10g/L，K + 0.2-0.5g/L，Mg 2+ 0.03-0.1g/L，Na + 0.2-0.8g/L，Fe 2+ (5-15)×10 -3 g/L, the balance being water, and the pH value is 6.5-8;
the carbon source is selected from one or more of glucose, starch, sucrose, dextrin, sodium succinate and sodium citrate;
the nitrogen source is selected from one or more of yeast extract powder, peptone, ammonium salt, urea and soybean meal powder.
6. A method of purifying a body of water comprising the step of inoculating the candida palmatine of claim 1 or 2 or the microbial inoculant of claim 3 into a body of water, the body of water being a farmed sewage/wastewater, the pH of the body of water being 4.0-8.0, the NaCl content of the body of water being no more than 25 per mill, the nitrite nitrogen concentration of the body of water being no more than 400mg/L.
7. Use of candida palmatina as claimed in claim 1 or 2 or the microbial agent of claim 3 for degrading ammonia nitrogen and nitrite nitrogen in water.
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| CN118109322B (en) * | 2024-03-04 | 2024-08-23 | 湘湖实验室(农业浙江省实验室) | Candida XHZG06-95A3 and application thereof |
| CN118240555A (en) * | 2024-03-11 | 2024-06-25 | 湘湖实验室(农业浙江省实验室) | Method for preparing acidified soil conditioner based on microbial fermentation |
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