A facultative autotrophic hydrogen oxidizing bacterium, its acquisition method and application

Abstract

The present invention provides a facultative autotrophic hydrogen oxidizing bacterium, an acquisition method, and an application, belonging to the field of microbial technology. The facultative autotrophic hydrogen oxidizing bacterium is a wild strain and has not undergone molecular genetic manipulation. Therefore, the single-cell protein product produced can be used for feed and food, without the risk of genetic modification; at the same time, compared with the mutagenic strain, the genetic stability is high. The facultative autotrophic hydrogen oxidizing bacterium can not only grow efficiently autotrophically using carbon dioxide, but also grow rapidly heterotrophically using high-concentration glucose, achieving carbon dioxide fixation and fermentation in glucose. The facultative autotrophic hydrogen oxidizing bacterium Q01 can efficiently produce single-cell protein in a mixotrophic manner, with a yield of up to 3.36g / l / h.

Description

Technical Field

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

[0002] Hydrogen oxidizing bacteria are a type of microorganism that can obtain energy by oxidizing hydrogen gas and grow heterotrophically on organic matter such as carbon dioxide or sugars. They are chemolithoautotrophic and are primarily found in soil. Hydrogen oxidizing bacteria can produce single-cell proteins, which do not occupy arable land and are not subject to climate restrictions. They have high production efficiency, high bacterial protein content, and a rich variety of amino acids. Compared to plant and animal proteins, they are both economically efficient and environmentally friendly. Their diverse substrate utilization spectrum also helps reduce production costs and ensure production stability.

[0003] At present, the main hydrogen-oxidizing bacteria producing single-cell protein are C. necator However, C. necator The strain has limited ability to utilize glucose, making it difficult to efficiently utilize cheap glucose wastewater such as molasses wastewater and cellulose hydrolysate to produce single-cell protein. C. necator H16 cannot utilize glucose and needs to be obtained through mutagenesis or genetic engineering strategies. However, it is often unstable and cannot be used in feed and food production, which limits its application range. At the same time, there are few wild-type glucose-utilizing strains of the same species, the fermentation delay period is long, the glucose concentration tolerance is lower than 25g / l, and the specific growth rate is low, only 0.24h -1 , see C. necator DSM 545, "Model of aceticacid-affected growth and poly(3-hydroxybutyrate) production by Copper-loving murderer DSM 545 and C. necator NCIMB 11599, "Recycling potential of Copper-loving murderer for life support in space: Production of SCPs from volatile fatty acid and urea mixture》.

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

[0005] Based on the above objectives, the present invention provides a facultative autotrophic hydrogen oxidizing bacteria, an acquisition method and an application.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention obtains a wild-type facultative autotrophic hydrogen oxidizing bacterium by enriching the garden soil microorganisms in an autotrophic environment and screening glucose. The facultative autotrophic hydrogen oxidizing bacterium is identified as Copperbacterium nematophilum ( Copper-loving murderer ), strain number Q01, was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on May 12, 2025, with the deposit number: CGMCC NO.34397.

[0008] In a second aspect, the present application provides a composition comprising the above-mentioned facultative autotrophic hydrogen oxidizing bacteria. To be applicable to different scenarios and environments, other materials in the composition are not limited to other bacterial species, reagents, etc.

[0009] In a third aspect, the present application provides a method for obtaining the above-mentioned facultative autotrophic hydrogen oxidizing bacteria, which specifically comprises:

[0010] S01: Place a soil sample in DSMZ Medium 81 liquid culture medium and incubate it in a sealed environment at 30°C in an oxygen-containing mixed gas environment. During the incubation process, the mixed gas is replenished in a timely manner and subculture is performed at an inoculation rate of 5%; wherein the soil sample is derived from garden soil, and the oxygen-containing mixed gas is H2, O2, and CO2 in a volume ratio of 6-9:1-3:1, preferably H2, O2, and CO2 in a volume ratio of 7:2:1.

[0011] S02: After 20 consecutive subcultures, the culture solution is spread and separated, and statically cultured in a sealed environment filled with the mixed gas for 3-4 days;

[0012] S03: After the static culture, the cells were transferred to DSMZ Medium 81 solid culture medium containing 1 g / L glucose and cultured at 30°C for 2-3 days.

[0013] S04: Select the 10 fastest-growing colonies 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;

[0014] S05: The fastest growing colony in DSMZ Medium 81 solid culture medium containing 10 g / L glucose was selected, transferred to DSMZ Medium 81 liquid culture medium, and then autotrophically amplified at 30°C in an oxygen-containing mixed gas to obtain facultative autotrophic hydrogen oxidizing bacteria Q01.

[0015] In a fourth aspect, the present application provides an application of a facultatively autotrophic hydrogen oxidizing bacterium for producing single-cell protein. The facultatively autotrophic hydrogen oxidizing bacterium can produce single-cell protein under autotrophic, heterotrophic, mixotrophic, or heterotrophic-autotrophic conditions. In addition, the facultatively autotrophic hydrogen oxidizing bacterium can also produce single-cell protein using high-density fermentation of glucose and / or an oxygen-containing mixed gas.

[0016] The conditions for producing single-cell protein by the above-mentioned facultative autotrophic hydrogen oxidizing bacteria are: DSMZ Medium 81 culture medium containing 0-40 g / l glucose and an oxygen-containing mixed gas, wherein the oxygen-containing mixed gas is H2, O2, and CO2 in a volume ratio of 0-9:1-3:0-1.

[0017] In a fifth aspect, the present application provides another application of facultative autotrophic hydrogen oxidizing bacteria, namely, for fixing carbon dioxide.

[0018] In a sixth aspect, the present application provides another application of facultative autotrophic hydrogen oxidizing bacteria, namely, for glucose fermentation.

[0019] The present invention has the following beneficial effects:

[0020] (1) In this application, a wild-type facultative autotrophic hydrogen oxidizing bacterium Q01 was cultured from garden soil. This strain belongs to the copper-greedy strain of Hookworm. This strain is a wild strain and has not been subjected to molecular genetic manipulation. Therefore, the single-cell protein product produced can be used for feed and food without the risk of genetic modification. At the same time, compared with mutagenic strains, it has high genetic stability.

[0021] (2) The facultative autotrophic hydrogen oxidizing bacteria Q01 can efficiently grow autotrophically using carbon dioxide to achieve carbon dioxide fixation. Compared with H16 at the same inoculation density, the lag time of facultative autotrophic hydrogen oxidizing bacteria Q01 was reduced by about 5 hours, the maximum specific growth rate increased by 66.54%, the biomass dry weight increased by about 45.92%, and the single-cell protein content reached 75.86%.

[0022] (3) The facultative autotrophic hydrogen oxidizing bacteria Q01 can also use high concentrations of glucose for rapid heterotrophic growth, and can tolerate glucose concentrations up to 40 g / l, achieving fermentation in glucose.

[0023] (4) When the facultative autotrophic hydrogen oxidizing bacteria Q01 grows heterotrophically, when the glucose concentration is 9 g / l, the delay time is only 1.6 hours, which is much lower than the 9 hours in the existing technology; at the same time, the maximum specific growth rate μ can reach 0.37h-1 About 0.24h, which is higher than the highest data in the existing technology -1 About 54.2%.

[0024] (5) When the facultative autotrophic hydrogen oxidizing bacteria Q01 grows heterotrophically, when the glucose concentration is 40 g / l, the lag period is less than 5 hours, the maximum specific growth rate is as high as 0.37, and the protein content in aerobic growth using glucose is as high as 65-76%.

[0025] (6) The facultative autotrophic hydrogen oxidizing bacteria Q01 can efficiently produce single-cell protein in a mixotrophic manner with a yield of up to 3.36 g / l / h. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The ML phylogenetic tree of the facultative autotrophic hydrogen oxidizing bacteria Q01 provided in the examples of this application;

[0027] Figure 2 The facultative autotrophic hydrogen oxidizing bacteria Q01 and C.nector H16 autotrophic growth comparison curve;

[0028] Figure 3 The facultative autotrophic hydrogen oxidizing bacteria Q01 and C.nector H16 heterotrophic growth comparison curve. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0030] Example 1: Acquisition of facultative autotrophic hydrogen oxidizing bacteria

[0031] The present invention provides a facultative autotrophic hydrogen oxidizing bacteria, and the method for obtaining the strain includes:

[0032] S101: Garden soil from the applicant's location was collected and placed in a sealed 550 ml infusion bottle. DSMZ Medium 81 liquid culture medium (from the German Collection of Microorganisms and Cell Cultures, Leibniz Institute) was added. The culture was incubated at 30°C in a sealed environment containing an oxygenated gas mixture of H2, O2, and CO2 in a volume ratio of 7:2:1. During the incubation process, the hydrogen content in the infusion bottle was monitored daily. If the gas content was insufficient, the gas mixture was replenished promptly and subcultured at a 5% inoculation rate.

[0033] S102: After 20 consecutive subcultures, the culture was spread onto DSMZ Medium 81 solid culture plates and then placed in a sealed environment containing an oxygenated gas mixture at 30°C for 4 days. The oxygenated gas mixture consisted of H2, O2, and CO2 in a volume ratio of 7:2:1.

[0034] S103: After the static culture is completed, the single colony on the DSMZ Medium 81 solid culture medium plate is transferred to the DSMZ Medium 81 solid culture medium plate containing 1 g / L glucose using a sterilized bamboo stick, and the culture is continued at 30°C for 3 days.

[0035] S104: Select the 10 fastest-growing colonies and name them Q01-Q10. Transfer colonies Q01-Q10 to DSMZ Medium 81 solid culture medium plates containing 5 g / L and 10 g / L glucose, respectively, for two rounds of streak screening.

[0036] S105: The fastest-growing bacterial colony on DSMZ Medium 81 solid medium containing 10 g / L glucose was selected and transferred to DSMZ Medium 81 liquid medium. The colony was then autotrophically expanded at 30°C in an oxygenated mixed gas atmosphere containing H2, O2, and CO2 in a volume ratio of 7:2:1 to obtain facultative autotrophic hydrogen oxidizing bacteria Q01.

[0037] Example 2: Identification of facultative autotrophic hydrogen oxidizing bacteria

[0038] The facultative autotrophic hydrogen oxidizing bacteria Q01 obtained in Example 1 was sequenced by 16s rDNA sequencing, and the model constructed by ML phylogenetic tree was compared with the model constructed by MEGA12 software. Copper-loving The inter-genus genetic distance matrix was analyzed to obtain the 16srDNA sequence shown in SEQ ID No. 1, Table 1 and Appendix Figure 1 .

[0039] Table 1: Q01 and Q02 constructed based on the p-distance model using MEGA12 software Copper-loving Inter-generic genetic distance matrix

[0040]

[0041] According to 16srDNA sequence, Table 1, Appendix Figure 1 It can be determined that the facultative autotrophic hydrogen oxidizing bacteria Q01 is Copperbacterium necrotica ( C. murderer )kind.

[0042] Example 3: Autotrophic growth of facultative autotrophic hydrogen oxidizing bacteria Q01

[0043] The facultative autotrophic hydrogen oxidizing bacteria Q01 was added to a 550 ml sealed infusion bottle containing 50 ml of fresh DSMZ Medium 81 liquid culture medium at an inoculum concentration of OD600 = 0.2 to form the experimental group. C.nector H16 was added to a 550 ml sealed infusion bottle containing 50 ml of fresh culture medium at an inoculum concentration of OD600 = 0.2 to form a control group. C.nector H16 is referred to as H16. The infusion bottles of the experimental and control groups were filled with a mixture of H2, O2, and CO2 in a volume ratio of 7:2:1, and two parallel incubations were set up. No gas was added to the infusion bottles during the growth period. The experimental and control groups were incubated at 30°C and 150 rpm. OD600 was measured at regular intervals. The growth curve was coupled using the Gompertz equation. After 36 hours, the biomass dry weight and single-cell protein content were measured. The results are shown in Table 2 and Appendix. Figure 2 .

[0044] Table 2: Autotrophic growth curve coupling results, biomass dry weight and single cell protein content

[0045]

[0046] From Table 2 and Appendix Figure 2 It can be seen that when the inoculation concentration is 0.2, the delay time of facultative autotrophic hydrogen oxidizing bacteria Q01 is 9.64 hours, which is about 5 hours shorter than that of H16; the maximum specific growth rate μ is 0.0889 h -1 , an increase of 66.54% over H16; the biomass dry weight was 1.43g / L, about 45.92% higher than H16; the single-cell protein content was 75.86%, which was equivalent to H16.

[0047] Example 4: Heterotrophic growth of facultative autotrophic hydrogen oxidizing bacteria Q01

[0048] The logarithmic phase of facultative autotrophic hydrogen oxidizing bacteria Q01 was inoculated into a flask containing 50 ml of fresh DSMZ Medium 81 liquid culture 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 were added to the culture medium 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 replicates were set up for each experimental group. C.nector H16 was added to a flask containing 50 ml of fresh culture medium at an inoculum concentration of OD600 = 0.1. 1 g / l glucose was added to the culture medium to form a control group. Three parallel experiments were set up for this control group. C.nector H16 is abbreviated as H16.

[0049] The five experimental groups and the control group were cultured at 30°C with shaking, and samples were taken at regular intervals to measure OD600. The growth curve was coupled using the Gompertz equation, and the biomass dry weight and single cell protein content were measured after 12 hours. The results are shown in Table 3 and the attached table. Figure 3 .

[0050] Table 3: Autotrophic growth curve coupling results, biomass dry weight and single cell protein content

[0051]

[0052] From Table 3 and Appendix Figure 3 It can be seen that H16 cannot grow heterotrophically on glucose, but the facultative autotrophic hydrogen oxidizing bacteria Q01 can grow heterotrophically on glucose and produce single-cell protein at a content of 65.31-76.17%. In addition, when the glucose concentration is 9g / l, the facultative autotrophic hydrogen oxidizing bacteria Q01 has a delay time of only 1.6 hours, far lower than the 9 hours in the existing technology; at the same time, the maximum specific growth rate μ can reach 0.37h -1 About 0.24h, which is higher than the highest data in the existing technology -1 About 54.2%.

[0053] Example 5: Fermentation of facultative autotrophic hydrogen oxidizing bacteria Q01

[0054] Facultative autotrophic hydrogen oxidizing bacteria Q01 were inoculated at an OD600 concentration of 0.2 into four 1.4-liter gas-circulating bubble column reactors. Fresh DSMZ Medium 81 liquid culture medium containing 40 g / l glucose was added to the first reactor, and a mixture of H2, O2, and CO2 at a volume ratio of 7:2:1 was introduced into the gas-circulating bubble column reactor for cultivation, forming a mixotrophic group. Fresh culture 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 hours of cultivation, the air was replaced with a mixture of H2, O2, and CO2 at a volume ratio of 7:2:1, forming a heterotrophic-autotrophic group. Fresh culture medium was added to the third reactor, and a mixture of H2, O2, and CO2 at a volume ratio of 7:2:1 was introduced into the gas-circulating bubble column reactor for cultivation, forming an autotrophic group. Fresh culture medium containing 40 g / L glucose was added to the fourth reactor. Air was aerated 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. The aeration rate of the mixed gas was set at 4 L / min, and the pH was adjusted to 7.0 using urea. After 24 hours, samples were taken to determine the biomass dry weight and single-cell protein content, as shown in Table 4.

[0055] Table 4: Comparison of fermentation processes

[0056]

[0057] As shown in Table 4, the mixotrophic group achieved a biomass dry weight of 80.78 g / L and a production efficiency of 3.36 g / L / h; the heterotrophic-autotrophic group achieved a biomass dry weight of 5.53 g / L and a production efficiency of 2.31 g / L / h; the autotrophic group achieved a biomass dry weight of 47.88 g / L and a biomass yield of 2.00 g / L / h; and the heterotrophic group achieved a biomass dry weight of 50.53 g / L and a biomass yield of 2.11 g / L / h. Furthermore, the single-cell protein content harvested under the four processes was comparable, all exceeding 70%. This indicates that the mixotrophic method achieves the highest single-cell protein production efficiency while also enabling significant carbon dioxide fixation.

[0058] The above demonstrates that the facultatively autotrophic hydrogen oxidizing bacteria Q01 provided in this example can both efficiently grow autotrophically using carbon dioxide and rapidly grow heterotrophically using high concentrations of glucose. Furthermore, this mixotrophic approach enables high-density, rapid fermentation of facultatively autotrophic hydrogen oxidizing bacteria Q01, with a bacterial yield of 3.36 g / l / h.

[0059] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A facultative autotrophic hydrogen oxidizing bacterium, characterized in that The facultative autotrophic hydrogen oxidizing bacteria is Copperbacterium necrotica ( Cupriavidus necator ), strain number Q01, was deposited in the General Microbiology Center of China Culture Collection Administration on May 12, 2025, with the deposit number: CGMCC NO.34397.

2. A composition, characterized in that The invention comprises the facultative autotrophic hydrogen oxidizing bacteria according to claim 1.

3. Use of the facultative autotrophic hydrogen oxidizing bacteria according to claim 1 or the composition according to claim 2 in producing single-cell protein.

4. The use according to claim 3, characterized in that The facultative autotrophic hydrogen oxidizing bacteria produce single cell protein under autotrophic, heterotrophic, mixotrophic, and heterotrophic-autotrophic conditions.

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

6. The facultative autotrophic hydrogen oxidizing bacteria according to claim 1 or the composition according to claim 2 is used for fixing carbon dioxide.

7. The facultative autotrophic hydrogen oxidizing bacteria according to claim 1 or the composition according to claim 2 is used for glucose fermentation.

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