Corynebacterium ruminicola, microbial inoculum and its application

Abstract

The present invention relates to the field of microorganisms, and particularly to Corynebacterium vitaeruminis, a microbial agent thereof, and their applications. The present invention provides Corynebacterium vitaeruminis with a preservation number of CGMCC No. 33059. The present invention provides a Corynebacterium vitaeruminis and applies it to whey wastewater to study its functions, filling the domestic research gap.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to Corynebacterium ruminihabitum, a microbial agent and its applications. Background Art

[0002] Corynebacterium refers to Gram-positive bacilli. It is named because one or both ends of its members are swollen into a rod shape. It has various morphologies, irregular arrangements, often in a palisade shape or V shape, etc.; the staining is uneven, and there are metachromatic granules with darker staining at both ends. It has no spores, and most strains are non-motile. It is aerobic or facultatively anaerobic, has special nutritional requirements, and the G + C molar content in DNA is 52-68%. The cell wall polysaccharide is mainly arabinose and galactose.

[0003] There have been many studies on Corynebacterium, mostly concentrated on the research of glutamic acid, lysine and valine, etc., such as the patent (CN201610515320.X) A Corynebacterium glutamicum and its applications, the patent (CN 202011285971.7) Corynebacterium glutamicum with high yield of glutamic acid and its applications; the patent (CN 201610800601.X) Method for fermenting and producing L-lysine and modified Corynebacterium, the patent (CN 201911370732.9) A Corynebacterium with high yield of valine and its construction method and applications, etc. However, there are few studies on Corynebacterium ruminihabitum. Hong Yongcong's research found that Corynebacterium ruminihabitum has good degradation ability for cypermethrin, while Wang Zhihong's research found that Corynebacterium ruminihabitum can secrete phytase, and other functions are still unclear. Summary of the Invention

[0004] In view of this, the present invention provides Corynebacterium ruminihabitum, a microbial agent and its applications. The present invention provides a Corynebacterium ruminihabitum and applies it to whey wastewater to study its functions and fill the domestic research gap.

[0005] In order to achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0006] The present invention provides Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ) with a preservation number of: CGMCC No. 33059.

[0007] In some embodiments of the present invention, the above-mentioned Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis

[0008] The present invention also provides a microbial inoculum, comprising: the above-mentioned Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ).

[0009] The present invention also provides the use of the above-mentioned Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ) and / or the above-mentioned microbial inoculum in treating whey wastewater.

[0010] In some embodiments of the present invention, in the above-mentioned use, the Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ) and / or the microbial inoculum reduce the pH value of the whey wastewater.

[0011] In some embodiments of the present invention, in the above-mentioned use, the Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ) and / or the microbial inoculum increase the suspended solid content of the whey wastewater.

[0012] The present invention also provides a method for treating whey wastewater, in which after inoculating the Corynebacterium ruminihabitum ( Corynebacterium vitaeruminis ) and / or the microbial inoculum, fermentation culture is carried out and then mixed with the whey wastewater.

[0013] In some embodiments of the present invention, in the above-mentioned method, the fermentation culture is carried out using a fermentation medium; the fermentation medium comprises: YPD medium.

[0014] In some embodiments of the present invention, in the above-mentioned method, the YPD medium comprises: glucose 2% (m / v), yeast powder 1.0% (m / v), peptone 2% (m / v) and agar powder 1.8% (m / v).

[0015] In some embodiments of the present invention, in the above-mentioned method, the inoculation amount of the inoculation is 1.0%, and the viable cell count is 5900×10 4 cfu / mL.

[0016] The Corynebacterium ruminihabitum isolated by the present invention produces lactic acid, can grow on both LB medium and YPD medium, does not produce protease, has good cell sedimentation performance, has a flocculation effect, the optimal addition amount is more than 7.5%, and can increase the suspended solid content when applied to whey wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0018] Figure 1Show the colony morphology of LBSW24-01;

[0019] Figure 2 Show the microscopic morphology of the colonies of LBSW24-01;

[0020] Figure 3 Show that the fermentation broth decolorizes potassium permanganate;

[0021] Figure 4 Show that Corynebacterium rumenicola settles for 10 minutes.

[0022] Biological deposit description

[0023] Biological material: LBSW24-01; Taxonomic name: Corynebacterium rumenicola ( Corynebacterium vitaeruminis ) was deposited on December 13, 2024, at the General Microbiology Center of the China National Committee for Culture Collection of Microorganisms; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 33059. Detailed implementation manners

[0024] The present invention discloses Corynebacterium rumenicola, a microbial agent and its applications.

[0025] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the said expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0026] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-limiting, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0027] It should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0028] The use of any and all examples or exemplary language such as "for example" or "including" herein is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0029] In addition, the numerical ranges and parameters used to define the present invention are approximate values. Here, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific numerical value or range.

[0030] The present invention independently screened and isolated a strain of Corynebacterium, which was identified as Corynebacterium ruminihabitans. Corynebacterium vitaeruminis It was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The strain name is LBSW24-01, and the deposit number is CGMCC No. 33059. The deposit date was December 13, 2024.

[0031] The deposited Corynebacterium ruminihabitans LBSW24-01 with the deposit number CGMCC No. 33059 has cells that are oval-shaped under the microscope, without spores, without flagella, and is a Gram-positive bacterium; the strain is cultured in a constant temperature incubator at 37°C for 24 hours, and can form smooth-surfaced, convex, and neatly-edged milky white colonies on LB solid medium.

[0032] The said Corynebacterium ruminihabitans LBSW24-01 contains the gene sequence shown in SEQ ID NO: 1.

[0033] The said Corynebacterium ruminihabitans can grow on both YPD medium and LB medium, but YPD medium is superior to LB medium.

[0034] The said Corynebacterium ruminihabitans ferments to produce lactic acid.

[0035] The said Corynebacterium ruminihabitans has good sedimentation performance and has a flocculation effect.

[0036] The said Corynebacterium ruminihabitans has no protease activity.

[0037] Applying this Corynebacterium ruminihabitans to whey wastewater with an addition amount of 1.0% (v / v) can significantly increase the content of suspended solids in the effluent, with an increase of more than 25%.

[0038] In Examples 1 to 4 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0039] The following further elaborates the present invention in conjunction with examples:

[0040] Example 1 Strain Isolation

[0041] Isolated from the whey wastewater of a sewage treatment plant in Boxing County, Binzhou City, Shandong Province. Take 10 mL of the wastewater and add it to 90 mL of sterile water with glass beads. Shake it in a shaker for 30 min, and then continue to dilute it step by step to 10^4 in a laminar flow hood. Take 100 μL and spread it on a YPD plate, and culture it in a constant temperature incubator at 37 °C for 24 hours. Pick monoclonal colonies with different morphological sizes in the laminar flow hood and transfer them to 15 mL of fresh YPD liquid medium, and culture them in a constant temperature incubator at 35 °C for 24 hours. Keep 3 glycerol tubes for each strain, and reserve the remaining bacterial liquid for future use.

[0042] Primary functional screening: Detect the pH of the obtained bacterial liquid. The detection method uses a pH electrode method, with YPD medium as the negative control. Select the strains with a pH lower than the negative control as candidate strains for the next experiment. A total of 3 acid-producing strains were isolated. Among them, strain JM4 can grow on both YPD medium and LB medium. Streak the screened strain JM4 on an LB plate and culture it in a constant temperature incubator at 37 °C for 24 h. It can form smooth, convex, and neatly edged milky white colonies. The colony morphology is as Figure 1 shown. Under the microscope, the cells are oval, without endospores and flagella. The microscopic morphology is as Figure 2 shown. Then send it to BGI for sequencing, and the sequence is shown in the gene sequence list SEQ ID NO:1. After BLAST alignment, the homology of this sequence with Corynebacterium vitaeruminis DSM 20294 reaches 99.93%, belonging to Corynebacterium vitaeruminis Corynebacterium tumidum, and it is named LBSW24-01.

[0043] Example 2 Screening of fermentation medium

[0044] The formula of LB medium is: sodium chloride 10 g / L, yeast powder 5 g / L, peptone 10 g / L, agar powder 18 g / L (solid), and the balance is distilled water, pH 6.8 - 7.0, sterilized at 121 °C for 20 min.

[0045] The formula of YPD medium is: glucose 20 g / L, yeast powder 10 g / L, peptone 20 g / L, agar powder 18 g / L (solid), and the balance is distilled water, sterilized at 121 °C for 20 min.

[0046] Activation of the strain: In the laminar flow hood, pick the strain with the preservation number CGMCC No. 33059 preserved on the slant and transfer it to 200 mL of LB liquid medium, and culture it in a shaking incubator at 37 °C and 150 rpm for 24 h;

[0047] Inoculation: The prepared liquid seeds were respectively inoculated into 200 mL of LB liquid medium and 200 mL of YPD liquid medium at an inoculation amount of 1% (v / v), and cultured in a shaking incubator at 37°C at 150 rpm for 12 h.

[0048] The colony numbers and the changes in suspended substances were respectively detected. The detection methods refer to the determination of total number of colonies in food microbiological examination in GB 4789.2 - 2016, the gravimetric method for the determination of suspended substances in water quality in GB 11901 - 1989, and the Kjeldahl method for the determination of crude protein in feed in GB / T 6432 - 2018.

[0049] Table 1 Medium optimization

[0050]

[0051] After detection, the colony number and the content of suspended substances of Corynebacterium rumini in the YPD medium were much higher than those in the LB medium. Therefore, the optimal fermentation medium for Corynebacterium rumini is the YPD medium.

[0052] Example 3 Functional identification of Corynebacterium rumini

[0053] 3.1 Determination of amino acid content

[0054] Previous studies have shown that corynebacteria produce amino acids or small - molecule organic acids. To verify the function of this strain, the amino acid content and pH change in the fermentation broth were detected. The detection steps were as follows: Take an appropriate amount of fermentation broth, filter it with filter paper, and take the filtrate for the detection of pH and amino acid content. Use YPD as a control. The detection method for amino acid content refers to the determination of total free amino acids in tea in GB / T 8314 - 2013, and the pH was measured by the pH electrode method.

[0055] Table 2 Determination of amino acid content

[0056]

[0057] After detection, the results are shown in Table 2. The free amino acid content in YPD is higher than that in the fermentation broth, indicating that amino acids are consumed during the fermentation process. The pH of the fermentation broth is lower than that of YPD, which also shows that H + , is produced during the fermentation process. At the same time, the fermentation broth can decolorize the potassium permanganate solution (as Figure 3 shown), indicating that lactic acid is produced during the fermentation process. After detection, the lactic acid content is 976 mg / L.

[0058] 3.2 Flocculation analysis

[0059] 3.2.1 Flocculation rate detection

[0060] In Example 2, it was found that Corynebacterium rumini had a good sedimentation effect (as Figure 4As shown in the figure, it is speculated that Corynebacterium rumenicola may have a flocculation effect. Therefore, the flocculation rate of the obtained bacterial liquid was detected, and the determination method was as follows: A 5 g / L kaolin suspension was prepared with kaolin (analytical pure) as a simulated suspended solid polluted water body. 100 mL of the simulated water sample was added to a 250 mL conical flask, and 2 mL of the bacterial liquid and 0.1 mL of the coagulant aid 5% (w / v) CaCl2 solution were added respectively. The pH was adjusted to 6.8 - 7.0 using NaOH and HCl, and mixed at 500 rpm for 5 minutes on a magnetic stirrer. At the same time, a simulated water sample added with YPD medium was used as a blank control under the same conditions. Then it was left to stand for 10 minutes, and 10 mL of the supernatant was aspirated. The absorbance of the supernatant of the test sample and the absorbance of the supernatant of the blank control solution were measured at 550 nm using a UV spectrophotometer, and the flocculation rate was calculated using the following formula:

[0061] Flocculation rate (%) = (B - A) × 100 / B

[0062] In the above formula,

[0063] A - Absorbance of the supernatant of the test sample;

[0064] B - Absorbance of the supernatant of the blank solution.

[0065] Table 3 Detection of the flocculability of Corynebacterium rumenicola

[0066]

[0067] After detection and analysis, Corynebacterium rumenicola has a certain flocculability, and the addition amount needs to be studied.

[0068] 3.2.2 Effect of the addition amount of the strain on flocculability

[0069] 100 mL of the simulated water sample was added to each of the 7 250 mL conical flasks, and 0 mL, 2 mL, 5 mL, 7.5 mL, 10 mL, 15 mL, and 20 mL of the fermentation broth of Corynebacterium rumenicola LBSW24 - 01 were added respectively. Correspondingly, 20 mL, 18 mL, 15 mL, 12.5 mL, 10 mL, 5 mL, and 0 mL of YPD medium were added. 0.12 mL of the coagulant aid 5% (w / v) CaCl2 solution was added to each flask. The pH was adjusted to 6.8 - 7.0 using NaOH and HCl, and mixed at 500 rpm for 5 minutes on a magnetic stirrer. Then it was left to stand for 10 minutes, and 10 mL of the supernatant was taken. The absorbance of the supernatant of the test sample was measured at 550 nm using a UV spectrophotometer, and the flocculation rate was calculated using the following formula:

[0070] Flocculation rate (%) = (B - A) × 100 / B

[0071] In the above formula,

[0072] A - Absorbance of the supernatant of the test sample

[0073] B - Absorbance of the supernatant of the blank solution

[0074] Table 4 Relationship between the addition amount of bacterial solution and the flocculation rate

[0075]

[0076] It can be seen from the table that in order to achieve the best flocculation effect of *Corynebacterium ruminicola*, the minimum addition amount of the bacterial solution is 7.5%.

[0077] 3.3 Detection of protease activity

[0078] To verify whether *Corynebacterium ruminicola* has the property of producing protease, the protease activity of the fermentation broth of *Corynebacterium ruminicola* was detected by the Folin - phenol method. The detection steps are as follows: 1) The control group (YPD) and the experimental group (fermentation broth of LBSW24 - 01) were respectively diluted 5 times, and 1 mL of the diluted solution was taken and pre - heated at 40 °C for 2 minutes; 2) 2 mL of trichloroacetic acid was added to the control group, and 1 mL of casein solution was added to the experimental group, shaken well, and incubated at 40 °C for 10 minutes; 3) 1 mL of casein solution was added to the control group, and 2 mL of trichloroacetic acid was added to the experimental group, shaken well, incubated at 40 °C for 10 minutes, and filtered; 4) 1 mL of the filtrate was taken respectively, 5 mL of sodium carbonate solution was added, and then 1 mL of Folin - phenol working solution was added, and the solution was placed in a 40 °C water bath for color development for 20 min; 5) The absorbance was measured at a wavelength of 680 nm with a spectrophotometer, and the amino acid content was found from the standard curve; The operations in 4) and 5) were carried out with water instead of the fermentation broth as the reference solution.

[0079] After detection, the protease activity of the fermentation broth of *Corynebacterium ruminicola* LBSW24 - 01 was 0 μ / mL. Therefore, this *Corynebacterium ruminicola* does not produce protease.

[0080] Table 5

[0081]

[0082] Example 4 Application of *Corynebacterium ruminicola* in whey wastewater

[0083] Take 2 500 - mL Erlenmeyer flasks, add 200 mL of whey wastewater; correspondingly add 2 mL of distilled water and the fermentation broth of LBSW24 - 01 respectively, seal with plastic wrap and place in a shaker, shake - culture at 150 rpm and 37 °C for 24 hours. After the fermentation is completed, detect the pH change. Take 50 mL for the determination of the suspended solid content, the filtrate is used to detect the COD, the remaining solution is placed in a centrifuge, centrifuged at 5000 rpm for 5 minutes, discard the supernatant, and the precipitate is placed in an oven at 105 °C and dried for 8 hours for standby, which is used for the determination of the crude protein content.

[0084] Table 6 Application of Corynebacterium ruminicola in whey wastewater

[0085]

[0086] The results are shown in Table 6. Adding Corynebacterium ruminicola can reduce the pH of whey wastewater and increase the suspended solid content, with the suspended solid content increasing by 25.29%.

[0087] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Ruminogenic Corynebacterium ( Corynebacterium vitaeruminis ), characterized in that, Its deposit number is: CGMCC No.33059.

2. A microbial agent, characterized in that: include: The rumen-inhabiting Corynebacterium according to claim 1 ( Corynebacterium vitaeruminis ).

3. The rumen-inhabiting Corynebacterium according to claim 1 ( Corynebacterium vitaeruminis ) and / or the use of the microbial agent as claimed in claim 2 in reducing the COD content in whey wastewater.

4. The rumen-inhabiting Corynebacterium according to claim 1 ( Corynebacterium vitaeruminis ) and / or the use of the microbial agent as claimed in claim 2 in reducing the pH value of whey wastewater.

5. The rumen-inhabiting Corynebacterium according to claim 1 ( Corynebacterium vitaeruminis ) and / or the use of the microbial agent as claimed in claim 2 in increasing the suspended matter content of whey wastewater.

6. A method for treating whey wastewater, characterized in that: The rumen-inhabiting Corynebacterium as claimed in claim 1 ( Corynebacterium vitaeruminis ) and / or after inoculation of the microbial agent as described in claim 2, fermentation culture, and mixing with whey wastewater.

7. The method according to claim 6, characterized in that The fermentation culture adopts a fermentation medium; the fermentation medium includes: YPD medium.

8. The method according to claim 7, characterized in that The YPD culture medium comprises: 2% (m / v) glucose, 1.0% (m / v) yeast powder, 2% (m / v) peptone and 1.8% (m / v) agar powder.

9. The method according to any one of claims 6 to 8, characterized in that The inoculation volume was 1.0% and the viable count was 5.9×10 7 cfu / mL.

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