Kluyveromyces marxianus BMK7 for protein production and biogas slurry purification and application of Kluyveromyces marxianus BMK7

Abstract

The invention relates to the technical field of environmental microorganisms and resource fermentation, and discloses a Kluyveromyces marxianus BMK7 for protein production and biogas slurry purification and application of the Kluyveromyces marxianus BMK7, the BMK7 is Kluyveromyces marxianus and is obtained through ultraviolet mutagenesis and hygromycin B adaptive evolution, the preservation number is CCTCC NO: M 20251050, and the Kluyveromyces marxianus BMK7 has good growth performance and genetic stability. The strain can be efficiently fermented under the condition that biogas slurry serves as a main substrate, ammonia nitrogen and chemical oxygen demand (COD) in the biogas slurry are remarkably reduced, and meanwhile high-proportion mycoprotein is synthesized. The thalli have strong flocculability and good sedimentation characteristics, and are beneficial to protein recovery. The method can be widely applied to treatment and resource utilization of sewage such as breeding wastewater, kitchen fermentation liquor and anaerobic effluent, and has a good industrialization prospect.

Description

Technical Field

[0001] The present invention belongs to the field of environmental microorganisms and resource-based fermentation technology, and specifically relates to Kluyveromyces marxianus BMK7 for protein production and biogas slurry purification and its application. Background Art

[0002] Single-cell protein (SCP) has become an important alternative to traditional protein feed due to its rapid growth rate, high nutritional value, and wide adaptability to a variety of substrates. SCP not only efficiently utilizes waste resources but also effectively reduces ecological burden, making it a sustainable protein source with great potential.

[0003] In the livestock and poultry farming process, biogas slurry, as a typical by-product, is rich in organic matter and ammonia nitrogen and has high potential for resource utilization. However, due to the complex composition of biogas slurry and the presence of various inhibitory substances, its efficient conversion and utilization still face many challenges. Yeast, as a widely used industrial microorganism, has demonstrated great advantages in the conversion of complex substrate resources with its excellent environmental tolerance and efficient protein synthesis ability. However, the growth and protein synthesis ability of yeast strains in complex substrates such as biogas slurry are usually limited. Therefore, it is necessary to improve their adaptability and protein synthesis ability through mutagenesis breeding to promote their application in biogas slurry resource utilization. Summary of the Invention

[0004] The present invention aims to provide a Kluyveromyces marxianus strain BMK7 obtained by ultraviolet mutagenesis and hygromycin B adaptive evolution. The deposit number of the Kluyveromyces marxianus strain is CCTCC NO: M 20251050.

[0005] Another object of the present invention is to provide the application of Kluyveromyces marxii in biogas slurry treatment. The strain of the present invention has the characteristics of flocculation and ammonia nitrogen recovery, and can effectively reduce the ammonia nitrogen and COD concentrations in pig farm wastewater while producing bacterial protein, providing theoretical support for the recycling and harmless treatment of agricultural waste.

[0006] In order to achieve the above object, the present invention adopts the following technical measures:

[0007] Obtaining Kluyveromyces marxianus BMK7:

[0008] The applicant obtained a Kluyveromyces marxianus strain that produces high-protein bacterial proteins in a biogas slurry-based culture medium from Xinjiang yogurt. The strain was then subjected to UV mutagenesis and hygromycin B adaptive evolution, and a comprehensive comparison of bacterial biomass and protein content. The strain was deposited with the China Center for Type Culture Collection on May 14, 2025, and is named Kluyveromyces marxianus BMK7. Its deposit number is CCTCC NO: M20251050. The address is Wuhan University, Wuhan, China.

[0009] Kluyveromyces marxii BMK7 is a Gram-negative bacterium with milky white, opaque colonies.

[0010] The protection content of the present invention also includes:

[0011] The invention relates to a fermentation broth of Kluyveromyces marxianus BMK7, wherein the fermentation broth contains live bacteria of Kluyveromyces marxianus BMK7.

[0012] A compound, wherein the active ingredient of the compound contains Kluyveromyces marxianus BMK7.

[0013] Application of Kluyveromyces marxianus BMK7, Kluyveromyces marxianus BMK7 fermentation broth and / or their compound in sewage treatment.

[0014] In the above application, preferably, the sewage is biogas slurry;

[0015] In the above-mentioned application, preferably, the effect of the sewage treatment is to reduce the ammonia nitrogen value and / or COD value of the sewage.

[0016] Application of Kluyveromyces marxianus BMK7, Kluyveromyces marxianus BMK7 fermentation broth and / or a compound thereof in producing bacterial protein by fermenting biogas slurry.

[0017] In the above application, preferably, the application process is to inoculate Kluyveromyces marxii BMK7 into biogas slurry for fermentation.

[0018] In the above application, preferably, glucose is added to the biogas slurry;

[0019] In the above application, preferably, the fermentation temperature is 27-43°C;

[0020] In the above application, preferably, the pH of the fermentation is 7-9;

[0021] In the above application, preferably, the inoculation amount of the bacterial strain during the fermentation is 2-10%.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention provides, for the first time, a yeast strain, Kluyveromyces marxii, that produces high levels of bacterial protein in biogas slurry and exhibits excellent ammonia nitrogen and COD removal efficiencies. This strain provides a high-quality seed strain for single-cell protein production and biogas slurry treatment. Optimized screening of piggery biogas slurry has provided an effective strain source for resourceful utilization of piggery biogas slurry.

[0024] At the same time, it was found through tests that the strain can still exert its biological functions under higher temperature and pH conditions, has environmental tolerance, and is suitable for industrial applications.

[0025] Whole genome sequencing of Kluyveromyces marxianus BMK7 showed that multiple flocculation-related genes (such as FLO5 and FLO9) had non-synonymous SNP mutations, which gave the bacteria strong flocculation and good sedimentation properties, facilitating protein recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The protein production of Kluyveromyces marxianus BMK7 at different temperatures.

[0027] Figure 2 The protein yield of Kluyveromyces marxianus BMK7 under different carbon sources.

[0028] Figure 3 The protein production of Kluyveromyces marxianus BMK7 under different carbon-nitrogen ratios.

[0029] Figure 4 The protein yield of Kluyveromyces marxii BMK7 at different pH values.

[0030] Figure 5 The protein yield of Kluyveromyces marxianus BMK7 at different initial inoculation amounts.

[0031] Figure 6 The bacterial protein production and ammonia nitrogen and COD removal efficiency of Kluyveromyces marxianus BMK7 under different optimal fermentation conditions. DETAILED DESCRIPTION

[0032] In the technical solutions of the present invention, the reagents, unless otherwise specified, were purchased from biochemical stores, and the technical solutions, unless otherwise specified, are conventional techniques in the art.

[0033] YPD medium: Weigh 20 g of peptone, 10 g of yeast extract powder, and 20 g of glucose, and add 1000 mL of pure water.

[0034] YPD solid medium: weigh 20 g of peptone, 10 g of yeast extract powder, 20 g of glucose, and 15 g of agar, and add 1000 mL of pure water.

[0035] Basic biogas slurry culture medium: After the biogas slurry is filtered through a 0.45 μm filter membrane, 20 g of glucose is added to 1000 mL of the filtrate.

[0036] Diluted biogas slurry culture medium: add 4 times the amount of clean water to the biogas slurry, stir, and allow to settle naturally for 1 week, then discard the precipitate. Centrifuge at 6000 r / min for 10 min, take the supernatant and filter it with a 0.45 μm filter membrane. Add 20 g of glucose to 1000 mL of the filtrate.

[0037] Example 1:

[0038] Isolation, Purification and Mutation Breeding of Kluyveromyces marxianus

[0039] 1. Sample Collection and Pretreatment

[0040] Weigh 10g of yogurt sample into a conical flask, add 90mL of sterile water, mix thoroughly, and incubate overnight at 27°C. The next day, filter the sample with filter paper, and perform a gradient dilution of the filtrate with sterile water in a clean bench.

[0041] 2. Enrichment of bacterial strains

[0042] Use a pipette to draw up 100 μL of the diluted solution and inoculate it onto YPD solid medium. Spread the solution evenly using a glass spreading rod. Incubate the medium in an inverted position at 27°C for 24 hours. After 24 hours, pick a single colony from the medium and inoculate it onto a fresh plate of YPD solid medium for streak isolation and purification. Incubate the plate in an inverted position at 27°C for 24 hours. Repeat this process three times to obtain pure colonies.

[0043] 3. Initial screening of biogas slurry-tolerant yeast

[0044] After purification and cultivation, a single colony was selected and propagated in liquid YPD medium, and then inoculated into diluted biogas slurry medium for spreading to observe whether there was colony growth.

[0045] Dilute the biogas slurry culture medium: Add 4 times the amount of water to the biogas slurry and stir. After natural sedimentation for 1 week, discard the precipitate. Centrifuge at 6000 rpm for 10 minutes, collect the supernatant, add 20g / L glucose and 15g / L agar, and sterilize at 115°C for 30 minutes. The indicators of the biogas slurry before dilution in this example are shown in the following table:

[0046]

[0047] The strains that can tolerate the diluted biogas slurry culture medium were selected for further screening.

[0048] 4. Rescreening of biogas slurry-tolerant yeast

[0049] The strains selected in the initial screening were inoculated into YPD liquid medium to prepare bacterial solution. Take the ultrafiltration biogas slurry, add 20g / L glucose and 15g / L agar and sterilize at 115℃ for 30min. Use a pipette to draw 100μL of bacterial solution and dilute it with sterile water for 10min. -3 Then, spread the solution onto solid biogas slurry culture medium and incubate in a 27°C incubator for 72 hours. Observe for colony growth and size, and select bacteria with large colonies that can tolerate the above-mentioned basic biogas slurry culture medium and store them for subsequent screening.

[0050] 5. Growth Curve Determination

[0051] The selected strains were concentrated at 1% (OD 600 =1.0) inoculated into YPD liquid medium, cultured at 27°C and 180 r / min for 24 h, and measured every 2 h. The sterilized medium without inoculation of bacterial liquid was used as a blank control to measure the OD at different culture times. 600 , select the fastest growing bacteria for UV mutagenesis.

[0052] OD of different strains at different growth times 600 The values ​​are shown in the table below, and MK1 is selected as the candidate strain:

[0053] 6. UV mutagenesis

[0054] The strain MK1 screened from the biogas slurry was revived for two generations using YPD medium as the starting strain. After culturing to the logarithmic phase, the strain was taken out and shaken with an oscillator and placed in a centrifuge. The supernatant was discarded after centrifugation at 8,000 r / min for 10 min. PBS buffer was added and diluted 10-fold to 10 -2 Set aside. Pipette 2mL of bacterial suspension from each tube and transfer it to a Φ35mm culture dish, and store it in a sterile environment for later use. Irradiate it at a distance of 30cm from a 30W UV lamp for 0, 3, 5, 7, 10, and 15 minutes, and then store the culture dish in a sterile operating table in a strict light-shielding manner for 12 hours to avoid the effect of photoreactivation on mutagenesis. Select the strain irradiated for 15 minutes for adaptive evolution. The different mutagenic strains obtained were measured for growth rate according to step 5, and the OD values ​​at different growth times were 600 The values ​​are shown in the table below. This table shows the results of some strains. The results show that MK15.2 bacteria is the next strain for adaptive evolution:

[0055]

[0056] 7. Adaptive evolution of hygromycin B

[0057] The mutant shape caused by simple UV mutagenesis has poor genetic stability, so it is combined with hygromycin B adaptive evolution to further stabilize and improve the strain's ability to synthesize bacterial protein.

[0058] First, determine the initial inhibitory concentration of the starting strain MK15.2, draw 200 μL of seed solution, inoculate into basal biogas slurry culture medium (10 ml) containing different concentration gradients of hygromycin B (0, 20, 30, 50, 70 mg / L), place it in a shaker at 27 ° C and 180 r / min for 48 h, and measure the OD of the bacterial solution. 600 The initial inhibitory concentration was determined to be 20 mg / L based on the concentration at which MK15.2 growth was inhibited. The culture medium was taken out every 96 h and the OD 600 To 1, aspirate 200 μL and transfer to a medium with a higher concentration of hygromycin B inhibitor until it reaches 70 mg / L, and separate different hygromycin B-adapted strains. Dilute the mutagenized bacterial solution with sterile water to OD 600 = 1, 200 μL was inoculated into 10 ml of basal biogas slurry medium and cultured in a constant temperature shaking incubator at 27°C and 180 rpm for 72 h. The mutant with the highest biomass and protein content was screened for BMK7, which was adapted to 70 mg / L hygromycin B and had good genetic stability.

[0059]

[0060] The obtained BMK7 and initial MK15.2 strains were cultured for 10 generations. Each generation consisted of 200 μL of the previous generation inoculated into a 10 ml basal biogas slurry medium. The culture was then incubated for 72 hours in a constant temperature shaking incubator at 27°C and 180 rpm to obtain fermentation broths of BMK7 and initial MK15.2. As shown in the table below, the biomass of BMK7 obtained after hygromycin B adaptation was more stable across generations than that of MK15.2. Furthermore, whole genome sequencing of the BMK7 strain revealed non-synonymous SNP mutations in multiple flocculation-related genes (such as FLO5 and FLO9). Compared with the wild-type MK1 fermentation broth, more flocs were visible to the naked eye. This SNP mutation confers stronger flocculability and good sedimentation properties to the BMK7 bacteria, facilitating protein recovery and making it suitable for industrialization.

[0061]

[0062] 8. Purification and identification of high-yield bacterial protein strains in biogas slurry

[0063] After the isolated strain was streaked multiple times to obtain a pure single colony, colony morphology was observed for biochemical identification. Genomic DNA was extracted and its ITSrDNA sequenced and analyzed. Comprehensive identification and analysis confirmed that the strain was a member of the genus Kluyveromyces marxianus. The bacterium was deposited with the China Center for Type Culture Collection on May 14, 2025, under the classification name Kluyveromyces marxianus BMK7, with the deposit number CCTCC NO: M20251050. The address is Wuhan University, Wuhan, China.

[0064] Example 2:

[0065] Optimal fermentation conditions for Kluyveromyces marxianus BMK7 in biogas slurry:

[0066] The biogas slurry used in this embodiment is the biogas slurry in Example 1.

[0067] 1. Optimal Temperature Selection Test for Kluyveromyces marxianus BMK7

[0068] The optimal growth temperature for the bacteria in biogas slurry was determined through selective experiments. A 2% (v / v) inoculum was transferred to a 250 mL Erlenmeyer flask containing 100 mL of biogas slurry culture medium (sterilized and supplemented with 20 g / L glucose). The culture was then incubated at 27°C, 37°C, and 42°C in a shaker at 180 rpm for 48 hours. The cells were harvested by centrifugation at 10,000 × g for 5 minutes, washed with purified water, and then oven-dried at 50°C before measurement of biomass and protein content.

[0069]

[0070] 2. Optimal Carbon Source Selection Experiment for Kluyveromyces marxianus BMK7

[0071] The optimal carbon source for the growth of this bacterium in biogas slurry was determined through selection experiments. Based on pretreated biogas slurry, glucose, sucrose, glycerol, sorbitol, and sodium acetate were added as carbon sources, adjusting the carbon-nitrogen ratio to that obtained when 20 g / L glucose was added. Sterilization was performed at 115°C for 30 minutes. A 2% inoculum of the mutagenic bacteria solution was inoculated, and the culture was shaken at 42°C and 180 rpm for 48 hours. Biomass and protein content were measured after oven drying at 50°C in triplicate.

[0072]

[0073] 3. Selection test of optimal carbon-nitrogen ratio for Kluyveromyces marxianus BMK7

[0074] A selection experiment was conducted to determine the optimal carbon-nitrogen ratio for growth in biogas slurry. A 2% (v / v) inoculum was transferred to a 250 mL Erlenmeyer flask containing 100 mL of biogas slurry medium. A C / N ratio gradient (1:1 to 10:1) was achieved by adjusting the amount of glucose added. Cultures were shaken at 42°C and 180 rpm for 48 hours. Cells were harvested by centrifugation at 10,000 × g for 5 minutes, and the medium was washed away by repeated centrifugation with purified water. Biomass and protein content were then measured in triplicate after drying in a 50°C oven.

[0075]

[0076] 4. Optimal pH selection test for Kluyveromyces marxianus BMK7

[0077] The optimal pH for growth of the bacteria in biogas slurry was determined through selection experiments. Fermentation was performed at five different initial pH values: pH 5, 6, 7, 8, and 9. A 2% inoculum of the mutagenic bacteria was inoculated, the carbon-nitrogen ratio was adjusted to a uniform value, and the culture was shaken at 42°C and 180 rpm. After 48 hours, samples were collected and oven-dried at 50°C. Biomass and protein synthesis levels were analyzed in triplicate.

[0078]

[0079] 5. Experimental study on the optimal inoculum size of Kluyveromyces marxianus BMK7

[0080] The optimal inoculum concentration for the growth of the bacteria in biogas slurry was selected by selection test, and the inoculum concentration was set to 2%, 4%, 6%, 8%, and 10% (seed liquid OD 600 =1), all other conditions remained the same, except for the carbon-nitrogen ratio, and the culture was performed at 42°C with shaking at 180 rpm. After 48 h, samples were collected and dried in a 50°C oven for analysis of bacterial biomass and protein synthesis levels. Three replicates were used.

[0081]

[0082] 6. Orthogonal test

[0083] We selected optimal experimental conditions and designed orthogonal experimental groups. We then combined these conditions to create a four-factor, three-level orthogonal experiment. During the experiment, we controlled the range of variation for each factor to identify the optimal combination of conditions. By comparing the changes in bacterial biomass in the biogas slurry culture medium under different combinations, we ultimately determined the optimal fermentation conditions to be: a temperature of 42°C, a pH of 8, a C:N ratio of 3, a bacterial slurry addition rate of 4%, and glucose as the optimal carbon source.

[0084]

[0085] The test results are shown in the figure below:

[0086]

[0087] Example 3:

[0088] Effective application of Kluyveromyces marxianus BMK7 in biogas slurry under optimal conditions

[0089] This example explored the effects of initial sugar addition and 24-hour segmented sugar addition on the fermentation process of biogas slurry in a 3L fermenter (1L of biogas slurry culture medium) by optimizing the feeding strategy. The biogas slurry culture medium used in this example was a different batch from that used in the strain screening process described above. It was obtained by adding 20g of glucose to 1000mL of randomly collected biogas slurry after filtering it through a 0.45μm membrane.

[0090] (1) A single BMK7 colony was inoculated into a 15 mL centrifuge tube containing 10 mL of YPD medium and cultured in a constant temperature shaker at 42°C and 150 rpm for 12 h to obtain a primary seed solution;

[0091] (2) Use sterile water to adjust the primary seed solution to OD600 = 1, and transfer 200 μL of the solution to a 500 mL shake flask containing 100 mL of biogas slurry culture medium. Incubate the flask at 42°C and 150 rpm for 48 h to obtain the secondary seed solution.

[0092] (3) The secondary seed solution was adjusted to OD600 = 1 and inoculated at 4% into a 3 L fermentor with a working volume of 1000 mL. Only 4% of 0.5 g / mL glucose was added at 24 h. The pH was adjusted to 8 by feeding every 12 h. The remaining parameters were controlled as follows: 42°C, 300 rpm, ventilation rate 1 min / L, and dissolved oxygen (DO) was controlled at 30%;

[0093] (4) After 120 h, the fermentation was stopped and samples were taken every 12 h to measure the bacterial biomass, protein content, glucose content in the biogas slurry, ammonia nitrogen concentration, and COD. The pH changes were dynamically monitored using a biosensor analyzer.

[0094] Since the inoculum size is small, the bacterial biomass in time period 0 is recorded as 0. The indicators in different time periods are as follows:

[0095]

Claims

1. A strain of Kluyveromyces marxii obtained by artificial mutagenesis ( Kluyveromyces marxianus ) BMK7, the deposit number of the Kluyveromyces marxianus is CCTCC NO: M 20251050.

2. The Kluyveromyces marxianus BMK7 fermentation broth according to claim 1, wherein the fermentation broth contains live Kluyveromyces marxianus BMK7 bacteria.

3. A compound, wherein the active ingredient of the compound contains Kluyveromyces marxianus BMK7.

4. Use of the Kluyveromyces marxianus BMK7 according to claim 1, the Kluyveromyces marxianus BMK7 fermentation broth according to claim 2, and / or the compound according to claim 3 in sewage treatment.

5. The use according to claim 4, wherein the sewage is biogas slurry.

6. The use according to claim 5, wherein the sewage treatment effect is to reduce the ammonia nitrogen value and / or COD value of the sewage.

7. Use of the Kluyveromyces marxianus BMK7 according to claim 1, the Kluyveromyces marxianus BMK7 fermentation broth according to claim 2, and / or the compound according to claim 3 in producing bacterial protein by fermenting biogas slurry.

8. The use according to claim 4 or claim 7, wherein the use process is to inoculate Kluyveromyces marxianus BMK7 into biogas slurry for fermentation.

9. The use according to claim 8, wherein glucose is added to the biogas slurry.

10. The use according to claim 8, wherein the fermentation temperature is 27-43°C.

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