Facultative autotrophic hydrogen oxidizing bacteria as well as acquisition method and application thereof

Abstract

The invention provides facultative autotrophic hydrogen oxidizing bacteria as well as an acquisition method and application thereof, and belongs to the technical field of microorganisms. The facultative autotrophic hydrogen oxidizing bacterium is a wild strain and is not subjected to molecular genetics operation, so that a produced single-cell protein product can be used for feed and eating and has no transgenic risk; meanwhile, compared with a mutant strain, the genetic stability is high. The facultative autotrophic hydrogen oxidizing bacteria can be used for efficient autotrophic growth by utilizing carbon dioxide and can also be used for rapid heterotrophic growth by utilizing high-concentration glucose, so that fixation of carbon dioxide and fermentation in glucose are realized. The facultative autotrophic hydrogen oxidizing bacterium Q01 can efficiently produce single-cell protein in a facultative mode, and the yield is as high as 3.36 g / l / h.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to a facultative autotrophic hydrogen-oxidizing bacterium, a method for obtaining the same, and an application thereof. Background Art

[0002] Hydrogen-oxidizing bacteria are a type of microorganism that can obtain energy by oxidizing hydrogen and grow heterotrophically using organic substances such as carbon dioxide or sugars. They belong to the chemolithoautotrophic type and are mainly distributed in soil. Hydrogen-oxidizing bacteria can produce single-cell protein, which does not occupy arable land, is not restricted by climate, has high production efficiency, high cell protein content, and rich amino acid types. Compared with plant and animal proteins, it has the characteristics of high economic efficiency and strong environmental protection attributes. Moreover, the diverse substrate utilization spectrum also helps to reduce production costs and ensure production stability.

[0003] Currently, the hydrogen-oxidizing bacteria for producing single-cell protein mainly consist of C. necator several different strains. However C. necator the ability of the strains to utilize glucose is limited, and it is difficult to efficiently utilize cheap glucose wastewater such as molasses wastewater and cellulose hydrolysate to produce single-cell protein. For example, the model strain C. necator H16 cannot utilize glucose and needs to obtain this trait through mutagenesis or genetic engineering strategies, which often have poor stability and cannot be used for feed and food production, resulting in limited application scope. At the same time, there are few wild-type glucose-utilizing strains of the same species, with a long fermentation lag phase, a glucose concentration tolerance lower than 25 g / l, and a low specific growth rate, only 0.24 h -1 ; for details, see C. necator DSM 545, "Model of aceticacid-affected growth and poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545" and C. necator NCIMB 11599, "Recycling potential of Cupriavidus necator for life support in space: Production of SCPs from volatile fatty acidand urea mixture".

[0004] In addition, the existing industrial fermentation methods for single-cell protein are usually pure heterotrophic or pure autotrophic, and it is difficult to combine the high production efficiency of heterotrophy and the high carbon reduction ability of autotrophy. Therefore, there is an urgent need to obtain wild-type hydrogen-oxidizing bacterial strains that can grow efficiently and facultatively using glucose and carbon dioxide. Summary of the Invention

[0005] For the above purposes, the present invention provides a facultative autotrophic hydrogen-oxidizing bacterium, a method for obtaining the same, and an application thereof.

[0006] To achieve the above purposes, the present invention adopts the following technical solutions: In the first aspect, the present application enriches the garden soil microorganisms in an autotrophic environment and screens them with glucose to obtain a wild-type facultative autotrophic hydrogen-oxidizing bacterium. After identification, this facultative autotrophic hydrogen-oxidizing bacterium is Cupriavidus necator ( Cupriavidus necator ), with the strain number Q01, which was deposited in the China General Microbiological Culture Collection Center (CGMCC) on May 12, 2025, and the deposit number: CGMCC NO. 34397.

[0007] In the second aspect, the present application provides a composition, which includes the above-mentioned facultative autotrophic hydrogen-oxidizing bacterium. To be applicable to different scenarios and environments, the other materials in this composition are not limited to other strains, reagents, etc.

[0008] In the third aspect, the present application provides a method for obtaining the above-mentioned facultative autotrophic hydrogen-oxidizing bacterium, which specifically includes: S01: Place the soil sample in DSMZ Medium 81 liquid medium, incubate it in a sealed environment with an oxygen-containing mixed gas at 30°C, and replenish the mixed gas in a timely manner during the incubation process and subculture it at an inoculation rate of 5%; among them, the soil sample is derived from garden soil, and the oxygen-containing mixed gas is H2, O2, CO2 with a volume ratio of 6-9:1-3:1, preferably H2, O2, CO2 with a volume ratio of 7:2:1.

[0009] S02: After continuous subculture 20 times, spread and separate the culture broth, and statically culture it in a sealed environment filled with the mixed gas for 3-4 days; S03: After the static culture is completed, transfer it to DSMZ Medium 81 solid medium containing 1 g / L glucose and continue to culture it at 30°C for 2-3 days; S04: Select the 10 colonies with the fastest growth rate, and transfer them to DSMZ Medium 81 solid medium containing 5 g / L and 10 g / L glucose respectively for two rounds of streak screening; S05: Select the colony with the fastest growth rate on DSMZ Medium 81 solid medium containing 10 g / L glucose, transfer it to DSMZ Medium 81 liquid medium, and perform autotrophic amplification in an oxygen-containing mixed gas at 30°C to obtain the facultative autotrophic hydrogen-oxidizing bacterium Q01.

[0010] Fourthly, the present application provides an application of facultative autotrophic hydrogen-oxidizing bacteria, namely, for producing single-cell protein. The facultative autotrophic hydrogen-oxidizing bacteria can produce single-cell protein under autotrophic, heterotrophic, mixotrophic, and heterotrophic-autotrophic conditions. Additionally, the facultative autotrophic hydrogen-oxidizing bacteria can also ferment glucose and / or an oxygen-containing mixed gas to produce single-cell protein at high density.

[0011] The conditions for the above facultative autotrophic hydrogen-oxidizing bacteria to produce single-cell protein are: DSMZ Medium 81 containing 0 - 40 g / l glucose and an oxygen-containing mixed gas, wherein the oxygen-containing mixed gas is H2, O2, and CO2 with a volume ratio of 0 - 9:1 - 3:0 - 1.

[0012] Fifthly, the present application provides another application of facultative autotrophic hydrogen-oxidizing bacteria, namely, for carbon dioxide fixation.

[0013] Sixthly, the present application provides another application of facultative autotrophic hydrogen-oxidizing bacteria, namely, for glucose fermentation.

[0014] The present invention has the following beneficial effects: (1) In the present application, a wild-type facultative autotrophic hydrogen-oxidizing bacterium Q01 is cultured from garden soil, and this strain is Cupriavidus necator. This strain is a wild strain without molecular genetics operations, so the produced single-cell protein product can be used for feed and food, without transgenic risks; at the same time, compared with mutagenized strains, it has high genetic stability.

[0015] (2) The facultative autotrophic hydrogen-oxidizing bacterium Q01 can grow autotrophically using carbon dioxide efficiently, achieving carbon dioxide fixation. Compared with H16 at the same inoculation density, the lag time of the facultative autotrophic hydrogen-oxidizing bacterium Q01 is reduced by about 5 hours, the maximum specific growth rate is increased by 66.54%, the biomass dry weight is increased by about 45.92%, and the single-cell protein content reaches 75.86%.

[0016] (3) The facultative autotrophic hydrogen-oxidizing bacterium Q01 can also grow heterotrophically rapidly using high-concentration glucose, tolerate a glucose concentration of up to 40 g / l, and achieve fermentation in glucose.

[0017] (4) When the facultative autotrophic hydrogen-oxidizing bacterium Q01 grows heterotrophically and the glucose concentration is 9 g / l, the lag time is only 1.6 hours, far lower than 9 hours in the prior art; at the same time, the maximum specific growth rate μ can reach about 0.37 h -1 or so, which is about 54.2% higher than the highest data of 0.24 h in the prior art. -1 about 54.2%.

[0018] (5)When the facultative autotrophic hydrogen-oxidizing bacterium Q01 grows heterotrophically, when the glucose concentration is 40 g / l, the lag phase is less than 5 hours, the maximum specific growth rate is as high as 0.37, and the protein content during aerobic growth using glucose is as high as 65-76%.

[0019] (6)The facultative autotrophic hydrogen-oxidizing bacterium Q01 can efficiently produce single-cell protein in the mixotrophic mode, and the productivity is greater than 3.36 g / l / h. Description of the Drawings

[0020] Figure 1 The ML phylogenetic tree of the facultative autotrophic hydrogen-oxidizing bacterium Q01 provided in the embodiment of the present application; Figure 2 For the facultative autotrophic hydrogen-oxidizing bacterium Q01 provided in the embodiment of the present application and C. nector The comparison curve of autotrophic growth of H16; Figure 3 For the facultative autotrophic hydrogen-oxidizing bacterium Q01 provided in the embodiment of the present application and C. nector The comparison curve of heterotrophic growth of H16. Detailed Embodiments

[0021] The technical solutions of the present invention will be further explained and illustrated below through specific embodiments.

[0022] Example 1: Obtaining of Facultative Autotrophic Hydrogen-Oxidizing Bacterium The embodiment of the present application provides a facultative autotrophic hydrogen-oxidizing bacterium, and the obtaining method of the strain includes: S101: Collect the garden soil at the location of the applicant, place the garden soil in a 550 ml sealed infusion bottle, add the DSMZ Medium 81 liquid medium preserved by the German Collection of Microorganisms and Cell Cultures, Leibniz Institute, and incubate in a sealed environment with an oxygen-containing mixed gas at 30 °C. The oxygen-containing mixed gas is H2, O2, and CO2 with a volume ratio of 7:2:1. During the incubation process, monitor the hydrogen content in the infusion bottle every day. When the gas content is insufficient, supplement the mixed gas in time and subculture according to an inoculation rate of 5%.

[0023] S102: After continuous subculture for 20 times, spread and separate the cultured bacterial liquid on the DSMZ Medium 81 solid medium plate, and then place it in a sealed environment with an oxygen-containing mixed gas and statically culture at 30 °C for 4 d. The oxygen-containing mixed gas is H2, O2, and CO2 with a volume ratio of 7:2:1.

[0024] S103: After the static culture is completed, use a sterilized bamboo stick to transfer the monoclonal colonies on the DSMZ Medium 81 solid medium plate to the DSMZ Medium 81 solid medium plate containing 1 g / L glucose, and continue to culture at 30 °C for 3 d.

[0025] S104: Select the 10 colonies with the fastest growth rate, and name them Q01 - Q10 respectively. Transfer the colonies Q01 - Q10 to the DSMZ Medium 81 solid medium plates containing 5 g / L and 10 g / L glucose respectively for two rounds of streak screening.

[0026] S105: Select the colony with the fastest growth on the DSMZ Medium 81 solid medium containing 10 g / L glucose, transfer it to the DSMZ Medium 81 liquid medium, and perform autotrophic amplification in an oxygen-containing mixed gas at 30 °C to obtain the facultative autotrophic hydrogen-oxidizing bacterium Q01. Among them, the oxygen-containing mixed gas is H2, O2, and CO2 with a volume ratio of 7:2:1.

[0027] Example 2: Identification of facultative autotrophic hydrogen-oxidizing bacterium Perform 16srDNA sequencing on the facultative autotrophic hydrogen-oxidizing bacterium Q01 obtained in Example 1. At the same time, use the MEGA12 software to analyze the model for constructing the ML phylogenetic tree and Cupriavidus the intergeneric genetic distance matrix, and obtain the 16srDNA sequence shown in SEQ ID No.1, Table 1 and appendix Figure 1 .

[0028] Table 1: Intergeneric genetic distance matrix of Q01 constructed based on the p-distance model using MEGA12 software and Cupriavidus intergeneric From the 16srDNA sequence, Table 1, appendix Figure 1 it can be determined that the facultative autotrophic hydrogen-oxidizing bacterium Q01 is Cupriavidus necator ( C. necator ) species.

[0029] Example 3: Autotrophic growth of facultative autotrophic hydrogen-oxidizing bacterium Q01 Add the facultative autotrophic hydrogen-oxidizing bacterium Q01 to a 550 ml sealed infusion bottle containing 50 ml of fresh DSMZ Medium 81 liquid medium at an inoculation concentration of OD600 = 0.2 to form an experimental group. Add C. nector H16 purchased from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures to a 550 ml sealed infusion bottle containing 50 ml of fresh medium at an inoculation concentration of OD600 = 0.2 to form a control group. The following C. nectorH16 is simply referred to as H16. The upper spaces in the infusion bottles of the experimental group and the control group are filled with a gas mixture of H2, O2, and CO2 with a volume ratio of 7:2:1. Two parallels are set respectively, and no additional gas is supplemented in the upper space during the growth period. The experimental group and the control group are incubated at 30 °C with a rotation speed of 150 rpm. Samples are taken regularly to measure OD600, and the growth curves are coupled using the Gompertz equation. After 36 hours, the dry biomass weight and the single-cell protein content are measured, obtaining Table 2 and Appendix Figure 2 .

[0030] Table 2: Results of coupled autotrophic growth curves, dry biomass weight, and single-cell protein content From Table 2 and Appendix Figure 2 it can be seen that when the inoculation concentration is 0.2, the lag time of the facultative autotrophic hydrogen-oxidizing bacterium Q01 is 9.64 hours, which is about 5 hours less than that of H16; the maximum specific growth rate μ is 0.0889 h -1 , which is 66.54% higher than that of H16; the dry biomass weight is 1.43 g / L, which is about 45.92% higher than that of H16; the single-cell protein content is 75.86%, which is comparable to that of H16.

[0031] Example 4: Heterotrophic growth of the facultative autotrophic hydrogen-oxidizing bacterium Q01 Take the facultative autotrophic hydrogen-oxidizing bacterium Q01 in the logarithmic phase and inoculate it into a flask containing 50 ml of fresh DSMZ Medium 81 liquid medium at an inoculation rate of OD600 = 0.1. 0 g / l, 1 g / l, 3 g / l, 9 g / l, and 40 g / l of glucose are added to the medium respectively to form five experimental groups: Q01-0 g / l, Q01-1 g / l, Q01-3 g / l, Q01-9 g / l, and Q01-40 g / l. Three parallels are set for each experimental group. The C. nector H16 purchased from the Leibniz Institute German Collection of Microorganisms and Cell Cultures is added to a flask containing 50 ml of fresh medium at an inoculation concentration of OD600 = 0.1. 1 g / l of glucose is added to the medium to form a control group. Three parallels are set for this control group. The following C. nector H16 is simply referred to as H16.

[0032] The five experimental groups and the control group are all cultured with shaking at 30 °C. Samples are taken regularly to measure OD600, and the growth curves are coupled using the Gompertz equation. After 12 hours, the dry biomass weight and the single-cell protein content are measured, obtaining Table 3 and Appendix Figure 3 .

[0033] Table 3: Results of coupled autotrophic growth curves, dry biomass weight, and single-cell protein content As shown in Table 3 and the appendix Figure 3 It can be seen that H16 cannot grow heterotrophically using glucose, but the facultative autotrophic hydrogen-oxidizing bacterium Q01 can grow heterotrophically using glucose, and the content of the produced single-cell protein reaches 65.31 - 76.17%. In addition, when the glucose concentration is 9 g / l, the lag time of the facultative autotrophic hydrogen-oxidizing bacterium Q01 is only 1.6 hours, which is much lower than the 9 hours in the prior art; meanwhile, the maximum specific growth rate μ can reach 0.37 h -1 or so, which is higher than the highest data of 0.24 h in the prior art -1 about 54.2%.

[0034] Example 5: Fermentation of the facultative autotrophic hydrogen-oxidizing bacterium Q01 The facultative autotrophic hydrogen-oxidizing bacterium Q01 was added to four 1.4-liter gas-circulating bubble column reactors at an inoculation concentration of OD600 = 0.2. Fresh DSMZ Medium 81 liquid medium containing 40 g / l glucose was added to the first reactor, and a mixed gas of H2, O2, and CO2 with a volume ratio of 7:2:1 was introduced into the gas-circulating bubble column reactor for cultivation to form a facultative group in the facultative cultivation mode. Fresh medium containing 40 g / l glucose was added to the second reactor, and air was introduced into the gas-circulating bubble column reactor for cultivation. After 12 h of cultivation, the air was replaced with a mixed gas of H2, O2, and CO2 with a volume ratio of 7:2:1 and cultivation was continued to form a heterotrophic-autotrophic group in the heterotrophic-autotrophic mode. Fresh medium was added to the third reactor, and a mixed gas of H2, O2, and CO2 with a volume ratio of 7:2:1 was introduced into the gas-circulating bubble column reactor for cultivation to form an autotrophic group in the autotrophic mode. In the fourth reactor, fresh medium containing 40 g / l glucose was added, and air was introduced into the gas-circulating bubble column reactor for cultivation, and 40 g / l glucose was added again after 12 hours to form a heterotrophic group. Among them, the ventilation rate of the mixed gas was set at 4 L / min, and urea was used to adjust the pH to maintain it at 7.0. After 24 hours, samples were taken to measure the dry biomass weight and the single-cell protein content, and Table 4 was obtained

[0035] Table 4: Comparison of fermentation processes As can be seen from Table 4, the dry biomass weight in the mixotrophic group reached 80.78 g / L, and the production efficiency reached 3.36 g / L / h; the dry biomass weight in the heterotrophic-autotrophic group reached 5.53 g / L, and the production efficiency reached 2.31 g / L / h; the dry biomass weight in the autotrophic group reached 47.88 g / L, and the biomass productivity reached 2.00 g / L / h; the dry biomass weight in the heterotrophic group reached 50.53 g / L, and the biomass productivity reached 2.11 g / L / h. At the same time, the content of single-cell protein harvested under the four processes was quite similar, all above 70%. This indicates that the mixotrophic mode can achieve the highest single-cell protein production efficiency and can also achieve a large amount of carbon dioxide fixation.

[0036] The above content shows that the facultative autotrophic hydrogen-oxidizing bacterium Q01 provided in the embodiment of the present application can not only grow autotrophically using carbon dioxide efficiently, but also grow heterotrophically rapidly using high-concentration glucose. At the same time, through the mixotrophic mode, high-density rapid fermentation of the facultative autotrophic hydrogen-oxidizing bacterium Q01 can be achieved, and the cell yield reaches 3.36 g / L / h.

[0037] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A facultative autotrophic hydrogen-oxidizing bacterium, characterized in that, The facultative autotrophic hydrogen-oxidizing bacterium is Cupriavidus necator ( Cupriavidus necator Cupriavidus necator ), with the strain number Q01, which was deposited in the China General Microbiological Culture Collection Center on May 12, 2025, and the deposit number is CGMCC NO. 34397.

2. A composition, characterized in that, including the facultative autotrophic hydrogen-oxidizing bacterium described in claim 1.

3. The method for obtaining facultative autotrophic hydrogen-oxidizing bacteria according to claim 1, characterized in that, Comprising: Placing the soil sample in DSMZ Medium 81 liquid medium, incubating in a sealed environment with an oxygen-containing mixed gas at 30 °C, timely supplementing the mixed gas during the incubation process, and subculturing at an inoculation rate of 5%; After continuous subculturing 20 times, the cultured bacterial solution is spread for separation and statically cultured in a sealed environment filled with the mixed gas for 3 - 4 days; After the static culture is completed, it is transferred to DSMZ Medium 81 solid medium containing 1 g / L glucose and continuously cultured at 30 °C for 2 - 3 days; Selecting the 10 colonies with the fastest growth, and sequentially transferring them to DSMZ Medium 81 solid medium containing 5 g / L and 10 g / L glucose for two rounds of streak screening; Selecting the colony with the fastest growth on DSMZ Medium 81 solid medium containing 10 g / L glucose, transferring it into DSMZ Medium 81 liquid medium, and performing autotrophic amplification in an oxygen-containing mixed gas at 30 °C to obtain the facultative autotrophic hydrogen-oxidizing bacterium Q01.

4. The method for obtaining facultative autotrophic hydrogen-oxidizing bacteria according to claim 3, characterized in that, The oxygen-containing mixed gas is H2, O2, CO2 with a volume ratio of 6 - 9:1 - 3:

1.

5. The method for obtaining facultative autotrophic hydrogen-oxidizing bacteria according to claim 3, characterized in that, The soil sample is derived from garden soil.

6. Use of the facultative autotrophic hydrogen-oxidizing bacterium described in claim 1 or the composition described in claim 2 in the production of single-cell protein.

7. The application according to claim 6, wherein The facultative autotrophic hydrogen-oxidizing bacterium produces single-cell protein under autotrophic, heterotrophic, mixotrophic, and heterotrophic-autotrophic conditions.

8. The application according to claim 6, characterized in that, The conditions for the facultative autotrophic hydrogen-oxidizing bacterium to produce single-cell protein are: DSMZ Medium 81 liquid medium containing 0 - 40 g / l glucose and an oxygen-containing mixed gas, wherein the oxygen-containing mixed gas is H2, O2, CO2 with a volume ratio of 0 - 9:1 - 3:0 - 1.

9. Use of the facultative autotrophic hydrogen-oxidizing bacterium described in claim 1 or the composition described in claim 2 for carbon dioxide fixation.

10. Use of the facultative autotrophic hydrogen-oxidizing bacterium described in claim 1 or the composition described in claim 2 for glucose fermentation.

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