A kind of fecal alkali-producing bacteria and its application in algae removal

Abstract

The present invention relates to a faecal alkaligenes, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC No.25996 and a deposit date of October 31, 2022. Its 16S rDNA sequence is shown in SEQ ID No:1. The faecal alkaligenes can effectively inhibit the growth of Microcystis aeruginosa in river and lake water. Experiments have confirmed that under a dosage of 500 ppm and room temperature, the faecal alkaligenes can effectively inhibit the growth of Microcystis aeruginosa in river and lake water. 8 cell / mL of Microcystis aeruginosa substrate concentration, the algal lysis rate was above 95% in 72h.

Description

Technical Field

[0001] The invention relates to alcaligenes faecalis, in particular to alcaligenes faecalis capable of inhibiting the growth of cyanobacteria in eutrophic water, a fermentation method thereof and a microbial agent containing the same, belonging to the technical field of environmental microorganisms. Background Art

[0002] With the rapid development of industry and agriculture in my country, more and more production and living wastewater is discharged into various rivers and lakes. These wastewaters often contain high concentrations of nitrogen and phosphorus nutrients. High-concentration nutrient wastewater flowing into rivers and lakes is likely to cause algae outbreaks and water blooms. Studies have shown that the main cause of water blooms is Microcystis aeruginosa among cyanobacteria. Algae outbreaks in water bodies will reduce the dissolved oxygen in the water body, thereby affecting the normal life of aquatic organisms. In severe cases, it can lead to the collapse of the entire aquatic ecosystem. At the same time, some algae will produce algal toxins during their growth, affecting the survival of other aquatic organisms and leading to insufficient food supply. In addition, there are direct and potential hazards to human health, such as an increase in the incidence of respiratory diseases. Therefore, water blooms are regarded as one of the serious ecosystem damage events.

[0003] Among the current solutions to this problem in the industry, chemical methods have low operating costs, are easy to operate, and are quick to take effect, but they are prone to induce drug resistance and may also cause secondary pollution and cause ecological imbalance. Although physical methods have less impact on the environment, they cannot fundamentally solve the problem and require a lot of manpower, material and financial resources. The cost of governance is high and the scope of application is limited. They are not suitable for open rivers. Biological methods do not produce secondary pollution and have always been regarded as the preferred solution for green governance, but they also have the problems of slow effect, being unsuitable for sudden algal blooms, and their biological safety needs to be verified.

[0004] In the 1970s, people found that there was a good correlation between the content of algae chlorophyll in eutrophic water and the number of algae-dissolving bacteria. The screening and application of algae-dissolving bacteria has become a relatively active research direction in the field of biological algae control. At present, the isolated bacteria with algae-dissolving effect mainly include Vibrio, Pseudomonas, Alteromonas, Bacillus and Coccus, etc. Their main targets include freshwater or marine algae such as cyanobacteria, dinoflagellates and diatoms, but the algae-dissolving effect of these bacteria is still not prominent enough, slow to take effect and unstable. Summary of the invention

[0005] In view of the shortcomings of existing algae-lytic bacteria, the present invention provides a faecal alkaligenes bacteria and its application in removing Microcystis aeruginosa in rivers and lakes and thus inhibiting algal blooms in rivers and lakes.

[0006] A faecalis bacteria (Alcaligenes faecalis) YZ118, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.25996, the deposit date is: October 31, 2022, its 16S rDNA sequence is shown in SEQ ID No: 1, unless otherwise specified, the faecalis bacteria described in the present invention refers to the YZ118 strain.

[0007] The present invention also claims protection for a microbial agent comprising the above-mentioned Alcaligenes faecalis.

[0008] The beneficial effects of the alkalinity producing bacteria provided by the present invention are as follows:

[0009] 1) It can effectively remove Microcystis aeruginosa in river and lake water. When the inoculation amount of bacterial suspension is 500ppm, the room temperature, the light intensity is 2000lux, the light-dark cycle is 10-12h:10-12h, and the pH is 7-8, the algae concentration does not exceed 1×10 8 cell / mL, the algicidal rate reached more than 95% at 72h;

[0010] 2) Salt tolerance: Experiments have shown that the strain is moderately salt tolerant and can grow in salinity conditions below 5%, but the most suitable salinity for growth and reproduction is below 4%;

[0011] 3) The microbial agent containing the alkalinity-producing bacteria is easy to produce, has low cost and high yield, and is suitable for promotion and application in the market.

[0012] The fermentation method of alcaligenes faecalis of the present invention comprises the following steps:

[0013] (1) Primary seed culture: Alcaligenes faecalis was inoculated into an enrichment medium under sterile conditions, and cultured at 28-32°C and 150-230 rpm for 12-36 h to obtain a primary seed culture solution;

[0014] (2) Secondary seed culture: inoculate the primary seed culture solution into the enrichment medium at an inoculum rate of 1-5 vol% under sterile conditions, and culture at 28-32° C. and 150-230 rpm for 12-36 h to obtain the secondary seed culture solution;

[0015] (3) Fermentation: After the fermentation medium in the fermentation tank is disinfected, the secondary seed culture solution obtained in step (2) is inoculated into the fermentation medium at an inoculation rate of 5-10 vol%, and the fermentation is carried out under the conditions of controlling the temperature to 28-32° C., the ventilation ratio to 1:(1-2) and the rotation speed to 150-230 rpm. During the fermentation process, the pH is adjusted to 6-7.5. When the dissolved oxygen begins to rise, the fermentation is stopped to obtain the fermentation liquid of alcaligenes faecalis.

[0016] Preferably, the composition of the enrichment medium is: peptone 5-15 g / L, yeast extract powder 3-8 g / L, sodium chloride 5-15 g / L, the solvent is water, and the pH is 6.5-8;

[0017] Preferably, the fermentation medium is composed of: 30-50 g / L carbon source, 15-30 g / L nitrogen source, PO4 3- 0.5-0.8g / L, Mg 2+ 0.01-0.03g / L, Mn 2+ 0.05-0.10g / L, Ca 2+ 0.1-0.3 g / L, the solvent is water, and pH=6.5-8.

[0018] Further preferably, the carbon source is selected from glucose or sucrose or a combination of the two, and the nitrogen source is selected from yeast extract powder or peptone or a combination of the two.

[0019] The ventilation ratio refers to the ratio of the volume of air introduced into the fermentation tank per minute to the total volume of the fermentation liquid.

[0020] Preferably, the PO4 3- The source is one of dipotassium hydrogen phosphate and potassium dihydrogen phosphate or a combination of the two, the Mg 2+ The source is one or more of magnesium sulfate, magnesium chloride and magnesium nitrate, and the Mn 2+ The source is one or more of manganese sulfate monohydrate, manganese nitrate, and manganese chloride, wherein the Ca 2+ The source is one or more of calcium chloride, calcium nitrate, and calcium hydrogen phosphate.

[0021] In actual application, the form of the product can be determined according to actual usage and storage needs. When a liquid product is needed, the fermentation liquid can be diluted to the required concentration and used directly. When a solid product is needed, the fermentation liquid can be centrifuged to obtain bacterial mud, and then freeze-drying technology is used to prepare solid bacterial powder.

[0022] The present invention also claims protection for a method for purifying river and lake water using the above-mentioned activation solution of Alcaligenes faecalis or a microbial agent containing Alcaligenes faecalis, comprising the step of applying the above-mentioned activation solution of Alcaligenes faecalis or a microbial agent containing the Alcaligenes faecalis to river and lake water.

[0023] Preferably, the application amount of the activation solution of Alcaligenes faecalis and the microbial agent is 100 ppm or more, more preferably 100-5000 ppm, further preferably 200-2000 ppm, and most preferably 500-1000 ppm.

[0024] Preferably, the temperature during the purification process is controlled at 10-45°C, preferably 28-32°C.

[0025] Preferably, the algae concentration in river and lake water does not exceed 1×10 8 cell / mL.

[0026] The present invention also claims to protect the use of the above-mentioned alcaligenes faecalis and a microbial agent containing the same in the field of river and lake water purification.

[0027] Preferably, the Alcaligenes faecalis and the microbial agent containing the Alcaligenes faecalis are used to remove Microcystis aeruginosa in river and lake water. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] Example 1: Screening and separation of strains

[0030] 1. Enrichment culture:

[0031] A water sample containing algae was collected from Tai'an, Shandong. 10 mL of the water sample was inoculated into a 250 mL headspace bottle containing 100 mL of enrichment liquid culture medium (10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 1 L of distilled water, pH = 7.0, sterilized at 121°C for 20 min), and the culture was shaken on a constant temperature shaker at 30°C and 200 r / min for one week to obtain the enrichment solution.

[0032] 2. Initial screening:

[0033] Prepare sterile water and dilute the enriched solution to 10 -3 , 10 -4 , 10 -5 and 10 -6times, and then spread each dilution on YZ solid selective medium (KH2PO4 1g, NH4Cl 0.8g, MgCl2·6H2O0.8g, CaCl2·2H2O 0.01g, FeCl3·6H2O 0.01g, MnCl2·4H2O 0.04g, Na2S·9H2O1.2g, beef extract 2g, peptone 10g, agar 20g, distilled water 1L, pH=7.0, sterilized at 121℃ for 20min), place the coated plate in a constant temperature incubator at 30℃ and culture until a single colony grows, pick out single colonies with different morphologies and transfer them to test tube slant culture medium, culture at 30℃ for about 48h, and then transfer to a 4℃ refrigerator for storage for later use.

[0034] A total of nine strains were isolated according to the above method and named YZ111, YZ112, YZ113, YZ114, YZ115, YZ116, YZ117, YZ118 and YZ119.

[0035] 3. Rescreening:

[0036] The nine strains were inoculated into activation medium (tryptone 10 g, yeast extract 5 g, sodium chloride 10 g, distilled water 1 L, pH = 7.0, sterilized at 121 ° C for 20 min), and cultured for 48 hours at 200 r / min and 30 ° C in a shaker to obtain activation solution. The activation solution of each strain was used to treat Microcystis aeruginosa to test the algae lysing effect. When the algae concentration was 1×10 6 cell / mL, the activation solution inoculation amount of each experimental group was 100ppm, and the treatment was carried out at room temperature, a light intensity of 2000lux, a light-dark cycle of 10-12h:10-12h, and a pH of 7-8. The algae lysis rate was calculated by measuring the algae chlorophyll.

[0037] Determination of chlorophyll a in Microcystis aeruginosa: Chlorophyll a was selected as the growth index of Microcystis aeruginosa in the algae-bacteria co-culture experiment. The main operations were as follows: a certain volume of algae solution (V1) was centrifuged at 5,000 rpm for 10 min at room temperature, the bacteria were collected and then repeatedly frozen and thawed for 3 times to completely lyse the cells to facilitate the extraction of chlorophyll a; resuspended with a certain volume of 95% ethanol (V2) and placed in a 4°C refrigerator overnight; centrifuged at 5,000 rpm for 10 min at room temperature, the absorbance of the supernatant was measured at wavelengths of 665 nm, 649 nm, and 750 nm, the chlorophyll a content was calculated, and the algae lysis rate was calculated based on the chlorophyll a content. The formula is as follows:

[0038] Chl-a(mg / L)=[(A 665 -A 750 )×13.7-(A 649 -A 750)×5.76]V2 / V1

[0039] The results are shown in Table 1.

[0040] Table 1 Algicidal rate of each strain screened

[0041] YZ111 YZ112 YZ113 YZ114 YZ115 YZ116 YZ117 YZ118 YZ119 24h 0％ 10.1％ 0％ 0％ 30％ 0％ 12％ 50％ 0％ 48h 0％ 11.2％ 0％ 0％ 37.2％ 1％ 19.3％ 89％ 0％ 72h 0％ 12％ 0％ 1％ 41.1％ 1.3％ 24.8％ 100％ 0％ 120h 0％ 13％ 0％ 5.3％ 53.6％ 0.7％ 26％ 100％ 3.2％

[0042] It can be seen from the data in Table 1 that compared with other strains, the YZ118 strain has a faster and better effect in removing Microcystis aeruginosa. Microcystis aeruginosa can be completely removed within 72 hours. The strain is re-inoculated into the nutrient medium to prepare the activation solution, which is then stored in a glycerol tube at -80°C.

[0043] Example 2. Identification of YZ118 strain:

[0044] 1. Experimental methods

[0045] 1.1 Extraction of bacterial genomic DNA

[0046] (1) Collect 1.0×10 9 (1ml bacterial solution OD 600 1-1.5) of bacterial culture, centrifuge at 12,000×g for 30 seconds, discard the supernatant, and resuspend the precipitate with 150 μl of Buffer S to which RNase A has been added.

[0047] (2) Add 20 μl of lysozyme storage solution, mix well, and let stand at room temperature for 5 min.

[0048] (3) Add 30 μl of 0.25 mol / L EDTA (pH 8.0), mix well, and place on ice for 5 min.

[0049] (4) Add 450 μl of Buffer GA, vortex for 15 seconds, and incubate in a 65°C water bath for 10 minutes.

[0050] (5) Add 400 μl of Buffer GB and 1 ml of Buffer DV (precooled at 4°C), mix vigorously, and centrifuge at 12,000 × g for 2 min.

[0051] (6) Discard the upper phase as much as possible and keep the interphase precipitate and the lower phase. Add 1 ml of 4°C pre-cooled Buffer DV, mix vigorously, and centrifuge at 12,000 × g for 2 min.

[0052] (7) Discard the upper phase and transfer the lower phase to a filter (the filter is placed in a 2 ml centrifuge tube) and centrifuge at 12,000 × g for 1 min.

[0053] (8) Discard the filter and add 400 μl of Buffer BV to the filtrate and mix well.

[0054] (9) Place the preparation tube in a 2 ml centrifuge tube, transfer the mixed solution in step 8 into the preparation tube, and centrifuge at 12,000 × g for 1 min.

[0055] (10) Discard the filtrate, return the preparation tube to the original 2 ml centrifuge tube, add 500 μl Buffer W1, and centrifuge at 12,000 × g for 1 min.

[0056] (11) Discard the filtrate, return the preparation tube to the original 2 ml centrifuge tube, add 700 μl Buffer W2, and centrifuge at 12,000 × g for 1 min.

[0057] (12) Wash once more with 700 μl Buffer W2 using the same method.

[0058] (13) Discard the filtrate, return the preparation tube to the original 2 ml centrifuge tube, and centrifuge at 12,000 × g for 1 min.

[0059] (14) Place the preparation tube in another clean 1.5 ml centrifuge tube, add 100-200 μl of Eluent or deionized water to the center of the silica membrane, let stand at room temperature for 1 min, and centrifuge at 12,000 × g for 1 min to elute the DNA.

[0060] 2. PCR amplification of bacterial genome

[0061] Table 2 PCR amplification primer design

[0062] Primer name sequence 27F 5-AGAGTTTGATCCTGGCTCAG-3 1492R 5-CTACGGCTACCTTGTTACGA-3

[0063] PCR amplification reaction system

[0064] Add the following ingredients to a 0.2 mL centrifuge tube:

[0065] Table 3 PCR amplification reaction system

[0066] Reagents volume Genomic DNA (20 ng / μl) 1.0μl <![CDATA[10×Buffer (containing 2.5 mmol / L Mg 2+ )]]> 5.0μl Taq polymerase (5u / μl) 0.5μl dNTP(10mM) 1.0μl 27F primer (10uM) 1.0μl 1492R primer (10uM) 1.0μl <![CDATA[ddH2O]]> 40.5μl Total volume 50.0μl

[0067] Mix by flicking gently, collect the droplets on the tube wall to the bottom of the tube by instant centrifugation, and perform PCR reaction on a PCR amplification instrument. The reaction parameters are shown in Table 4:

[0068] Table 4 PCR amplification reaction program

[0069] Pre-denaturation transsexual annealing extend Final extension Number of cycles 95℃,5min 95℃，30s 57℃，30s 72℃, 1min30s 72℃,7min 35

[0070] After the reaction was completed, 3 μl of the PCR product was taken for 1% agarose gel electrophoresis, and the size of the PCR amplified fragment was confirmed based on the DNA marker band.

[0071] 3. Recovery of PCR products

[0072] The PCR product was recovered using the TIANGEN DNA purification and recovery kit. The specific operation was carried out according to the kit instructions. The steps are as follows:

[0073] (1) Cut the agarose gel containing the target DNA fragment under ultraviolet light and place it in a clean centrifuge tube. Weigh the gel.

[0074] (2) Add Buffer DE-A in a volume twice the weight of the gel, mix well, and heat at 65°C until the gel is completely melted.

[0075] (3) Add 0.5 volume of Buffer DE-B to Buffer DE-A and mix well.

[0076] (4) Transfer the mixture to a DNA preparation tube and centrifuge at 12,000 × g for 1 min. Discard the filtrate.

[0077] (5) Place the preparation tube back into a 2 ml centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12,000 × g for 30 s, and discard the filtrate.

[0078] (6) Place the preparation tube back into a 2 ml centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12,000 × g for 30 s, and discard the filtrate. Use the same method to add 700 μl of Buffer W2 and centrifuge at 12,000 × g for 1 min.

[0079] (7) Place the prepared tube back into a 2 ml centrifuge tube and centrifuge at 12,000 × g for 1 min.

[0080] (8) Place the preparation tube in a clean 1.5 ml centrifuge tube (provided in the kit), add 30-50 μl of deionized water to the center of the preparation membrane, and let it stand at room temperature for 1-2 minutes. Centrifuge at 12,000×g for 1 minute to elute the DNA.

[0081] 4. Sequence determination and analysis

[0082] The purified PCR product was taken and the 16S rDNA gene sequence was determined using a sequencer ABI3730-XL. The result of the determination is shown in SEQ ID No: 1.

[0083] 5. Sequence analysis

[0084] The full-length sequence obtained by splicing the two ends of the sequence was submitted to the NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) database for comparison. The similarity higher than 97% was considered to be the same species and the species identification result was Alcaligenes faecalis.

[0085] Example 3: Study on the tolerance and growth conditions of Alcaligenes faecalis

[0086] 1. Determination of the optimal carbon source for strain growth

[0087] Experimental method: LB medium (tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1L, pH = 7.0, 121℃ sterilization for 20min) was used as the basic medium, and 2.5g glucose, 2.38g sucrose and 3.33g sodium acetate were added to 100mL of the basic medium as carbon sources. Another experimental group cultured in pure LB medium was set up as the control group. After inoculation of YZ118 strain, they were cultured in a shaker at 30℃ and 180r / min, and their OD was measured regularly. 600 (OD 600 It indicates the absorbance of the solution at a wavelength of 600nm. This value is used to measure the concentration of bacterial culture solution. It is usually used to refer to the bacterial cell density. The growth of bacteria can be measured by OD 600 The growth degree of the bacteria was determined by monitoring the value of ) and the number of viable bacteria. The results are shown in Table 5.

[0088] Table 5 OD of YZ118 strain cultured in medium with different carbon sources 600 Value and viable count

[0089]

[0090] From the data in Table 5, it can be seen that the growth degree of the YZ118 strain is best when the carbon source in the culture medium is sucrose, so sucrose is determined to be the optimal carbon source for Alcaligenes faecalis.

[0091] 2. Determination of the optimal nitrogen source for strain growth

[0092] Experimental method: LB medium (tryptone 10g, yeast extract powder 5g, sodium chloride 10g, distilled water 1L, pH = 7.0, sterilized at 121℃ for 20min) was used as the basal medium. Yeast extract powder 11.1g, peptone 7.69g and urea 2.13g were added to 100mL of the basal medium as nitrogen sources. Another experimental group cultured in pure LB medium was set up as a control group. The YZ118 strain was inoculated and cultured in a shaker at 30℃ and 180r / min. The OD was measured regularly. 600The growth degree of the strain was determined by the value and the number of viable bacteria. The results are shown in Table 6.

[0093] Table 6 OD of YZ118 strain cultured in medium with different nitrogen sources 600 Value and viable count

[0094]

[0095]

[0096] From the data in Table 6, it can be seen that the growth of YZ118 strain is best when the nitrogen source in the culture medium is yeast extract powder, so yeast extract powder is determined to be the optimal nitrogen source for Alcaligenes faecalis.

[0097] 3. Determination of the optimal carbon-nitrogen ratio for strain growth

[0098] Experimental method: LB medium (10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 1 L of distilled water, pH = 7.0, sterilized at 121 ° C for 20 min) was used as the basal medium, sucrose was used as the carbon source, and yeast extract powder was used as the nitrogen source. 1.23 g of yeast extract powder was added to 100 mL of the basal medium, and 0.45, 0.57 and 0.68 g of sucrose were added respectively to obtain three groups of experimental culture media with C:N = 8:1, C:N = 10:1, and C:N = 12:1 to determine the optimal carbon-nitrogen ratio. Another group of LB culture media without additional carbon and nitrogen sources was set up as a blank control group, and the YZ118 strain was inoculated and cultured in a shaker at 30 ° C, 180 r / min, and its OD was measured regularly. 600 The growth degree of the bacteria was determined by the value and the number of viable bacteria.

[0099] Table 7 OD of YZ118 strain cultured in medium with different carbon-nitrogen ratios 600 Value and viable count

[0100]

[0101] It can be seen from the data in Table 7 that the growth rate of strain YZ118 is best when the carbon-nitrogen ratio of the culture medium is 10:1, so the carbon-nitrogen ratio of 10:1 is the optimal growth carbon-nitrogen ratio for this strain.

[0102] 4. Determination of the optimal salinity for strain growth

[0103] Experimental method: LB medium (tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1L, pH = 7.0, sterilized at 121℃ for 20min) was used as the basal medium. A group of experimental groups without additional salinity was set up as a blank control. On the basis of LB medium, the other experimental groups added additional NaCl 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g to 100mL of the medium and set the salinity gradient to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%. After inoculation with YZ118 strain, they were cultured in a shaker at 30℃, 180r / min, and their OD was measured regularly. 600 The growth degree of the bacteria was determined by the value and the number of viable bacteria.

[0104] Table 8 OD of YZ118 strain after culture in medium with different salinities 600 Value and viable count

[0105]

[0106] As can be seen from Table 8, strain YZ118 is moderately salt-tolerant and can grow at salinity below 5%, but its growth condition is optimal when the salinity of the culture medium is below 4%.

[0107] Example 4: Production and post-processing process of alcaligenes faecalis

[0108] 1. Strain fermentation

[0109] 1) Primary shake flask activation

[0110] In a sterile environment, pick one ring of Alcaligenes faecalis YZ118 strain and inoculate it into a 250mL Erlenmeyer flask filled with 100mL enrichment medium (10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, 1L of distilled water, pH=7.0, sterilized at 121℃ for 20min), and culture it at 30℃ and 180r / min for 24h to obtain a primary activation solution.

[0111] 2) Secondary shake flask culture

[0112] In a sterile environment, transfer 10 mL of the primary activation solution to four 1L Erlenmeyer flasks containing 500 mL of enriched culture medium (10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 1 L of distilled water, pH = 7.0, sterilized at 121°C for 20 min), and culture at 30°C and 180 r / min for 24 h to obtain the secondary activation solution.

[0113] 3) 1t tank fermentation culture

[0114] The fermentation medium was sterilized at 121°C for 20 min. The formula of the fermentation medium was: sucrose 40 g / L, yeast extract 20 g / L, MgSO4 0.1 g / L, MnSO4 0.2 g / L, CaCl2 0.57 g / L, K2HPO4 1.2 g / L. The secondary seed culture solution was inoculated into the fermentation medium at an inoculation rate of 5-10 vol%. The fermentation tank was filled with 70% of the volume. The initial pH was adjusted to 7.0. The aeration ratio was 1:1.25 (m 3 ·min / m 3 ), 180rpm, 30 ℃ fermentation culture, during the fermentation process by adding ammonia to control pH = 6.5, the fermentation cycle is about 24h, when the dissolved oxygen begins to rise, the fermentation is stopped immediately, at this time the fermentation is in the late logarithmic phase, the number of viable bacteria is as high as 30 billion cfu / mL, the bacterial activity is the strongest, the fermentation nutrient residues are the least, the number of viable bacteria decay is less during storage.

[0115] 2. Post-treatment process of fermentation broth

[0116] In order to obtain bacterial powder, the fermentation broth can be treated by a post-treatment process of freeze drying, so that the amount of live bacteria can be better maintained. The preferred post-treatment process is freeze drying.

[0117] Example 5: Evaluation of the application of Alcaligenes faecalis in contaminated water bodies containing Microcystis aeruginosa

[0118] The purified YZ118 strain was inoculated into a nutrient liquid culture medium (10 g of tryptone, 5 g of yeast extract powder, 10 g of sodium chloride, 1 L of distilled water, pH = 7.0, sterilized at 121°C for 20 min), and cultured on a shaker at 30°C and 180 r / min for 2 days to prepare a bacterial suspension. The prepared bacterial suspension was then applied to a 250 mL headspace bottle containing contaminated water for an experiment. The wastewater contained different algae concentrations.

[0119] The experiment was arranged as follows:

[0120] Experimental group: Inoculate 500ppm bacterial suspension into polluted water with different algae concentrations. Each experimental group is set up in three parallels, and each experimental group is set up as a control group with polluted water without bacterial suspension inoculation. The treatment is carried out at room temperature, light intensity of 2000lux, light-dark cycle of 10-12h:10-12h, pH=7-8, and the algae lysis rate is calculated by measuring chlorophyll a. The experimental groups are arranged as follows:

[0121] Experimental group 1: algae concentration 1×10 8 cell / mL;

[0122] Experimental group 2: algae concentration 5×10 7 cell / mL;

[0123] Experimental group 3: algae concentration 1×10 7 cell / mL;

[0124] Determination of chlorophyll a in Microcystis aeruginosa: Chlorophyll a was selected as the growth index of Microcystis aeruginosa in the algae-bacteria co-culture experiment. The main operations were as follows: a certain volume of algae solution (V1) was centrifuged at 5,000 rpm for 10 min at room temperature, the bacteria were collected and then repeatedly frozen and thawed for 3 times to completely lyse the cells to facilitate the extraction of chlorophyll a; resuspended with a certain volume of 95% ethanol (V2) and placed in a 4°C refrigerator overnight; centrifuged at 5,000 rpm for 10 min at room temperature, the absorbance of the supernatant was measured at wavelengths of 665 nm, 649 nm, and 750 nm, the chlorophyll a content was calculated, and the algae lysis rate was calculated based on the chlorophyll a content. The formula is as follows:

[0125] Chl-a(mg / L)=[(A 665 -A 750 )×13.7-(A 649 -A 750 )×5.76]V2 / V1

[0126] The results are shown in Table 9.

[0127] Table 9 Algae lysis rate of each experimental group over time

[0128] Measuring time Control group 1 Experimental Group 1 Control group 2 Experimental Group 2 Control group 3 Experimental Group 3 24h 3.0％ 63.9％ 2％ 70.5％ 3.1％ 59.1％ 48h 8.1％ 72.5％ 5.2％ 78.5％ 6.0％ 85.4％ 72h 9.5％ 90.0％ 7.3％ 92％ 6.5％ 95％

[0129] As shown in Table 9, the YZ118 strain has a significant effect on the removal of algae in polluted water. It shows a strong algae removal effect in wastewater containing different algae concentrations. 8 When the algae concentration was below 1 cell / mL, the algae lysis rate reached over 90% in 72 hours.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A faecal alkaligenes ( Alcaligenes faecalis ), characterized in that It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the accession number CGMCC No.25996.

2. A microbial agent, characterized in that: The active ingredient comprises the Alcaligenes faecalis according to claim 1.

3. The fermentation method of Alcaligenes faecalis according to claim 1, characterized in that: The steps include: (1) Primary seed culture: Alcaligenes faecalis is inoculated into an enrichment medium under sterile conditions, and cultured at 28-32°C and 150-230 rpm for 12-30 h to obtain a primary seed culture solution; (2) Secondary seed culture: Under sterile conditions, the primary seed culture solution is inoculated into an enrichment medium at an inoculum rate of 1-5 vol%, and cultured at 28-32° C. and 150-230 rpm for 12-36 h to obtain a secondary seed culture solution; (3) Fermentation: After the fermentation medium in the fermentation tank is disinfected, the secondary seed culture solution obtained in step (2) is inoculated into the fermentation medium at an inoculation rate of 5-10 vol%, wherein the carbon source in the fermentation medium is selected from one of glucose or sucrose or a combination of the two, and the nitrogen source is selected from one of yeast extract powder or peptone or a combination of the two. The fermentation is carried out under the conditions of controlling the temperature to 28-32° C., the ventilation ratio to 1:(1-2) and the rotation speed to 150-230 rpm. During the fermentation process, the pH is adjusted to 6-7.

5. When the dissolved oxygen begins to rise, the fermentation is stopped to obtain the fermentation liquid of alcaligenes faecalis.

4. The fermentation method according to claim 3, characterized in that The enrichment medium is composed of: 5-15 g / L peptone, 3-8 g / L yeast extract powder, 5-15 g / L sodium chloride, the solvent is water, and the pH is 6.5-8; The fermentation medium is composed of: 30-50 g / L carbon source, 10-30 g / L nitrogen source, PO4 3- 0.5-0.8 g / L, Mg 2+ 0.01-0.03 g / L, Mn 2+ 0.05-0.10g / L, Ca 2+ 0.1-0.3 g / L, the solvent is water, and pH=6.5-8.

5. A method for purifying river and lake water, comprising the step of applying the activation solution of Alcaligenes faecalis according to claim 1 or the microbial agent according to claim 2 to the river and lake water, wherein the application amount of the activation solution of Alcaligenes faecalis and the microbial agent is more than 100 ppm, and is used to remove Microcystis aeruginosa in river and lake water.

6. The method according to claim 5, characterized in that The application amount of the activation solution of alcaligenes faecalis and the microbial agent is 100-5000ppm.

7. The method according to claim 6, characterized in that The application amount of the activation solution of alcaligenes faecalis and the microbial agent is 200-2000ppm.

8. The method according to claim 7, characterized in that The application amount of the activation solution of alcaligenes faecalis and the microbial agent is 500-1000ppm.

9. The method according to any one of claims 5 to 8, characterized in that: The temperature is controlled at 10-45°C during the purification process.

10. The method according to claim 9, characterized in that The temperature during the purification process is controlled at 28-32°C.

11. The method according to any one of claims 5 to 8 and 10, characterized in that: The algae concentration in the river and lake water shall not exceed 1×10 8 cell / mL.

12. The method according to claim 9, characterized in that The algae concentration in the river and lake water shall not exceed 1×10 8 cell / mL.

13. Use of the alcaligenes faecalis described in claim 1 and the microbial agent described in claim 2 in the field of river and lake water purification for removing Microcystis aeruginosa in river and lake water.

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