Multi-stage gradient circulating culture method of clostridium ethanolicum

Abstract

The invention provides a multistage gradient circulating culture method of clostridium ethanolicum, and relates to the technical field of clostridium ethanolicum culture. The method is carried out under anaerobic and sterile conditions, and comprises the following steps: obtaining clostridium ethanolicum glycerol bacteria; the method comprises the following steps: resuscitating glycerol bacteria of clostridium ethanolicum with high resuscitation survival rate to obtain clostridium ethanolicum liquid, inoculating the clostridium ethanolicum liquid into a proliferation culture medium for proliferation culture, and sequentially inoculating the clostridium ethanolicum liquid subjected to proliferation culture into a multi-stage reactor for multiple times of propagation and domestication to obtain domesticated clostridium ethanolicum. According to the method, a small amount of ethanol clostridium glycerol bacteria are used as raw materials for resuscitation, and the resuscitated ethanol clostridium is gradually subjected to expanding culture and domestication in a sterile anaerobic environment, so that the growth of infectious microbes is controlled from the source, the resuscitation rate is high, meanwhile, the yield and the metabolic capability of the ethanol clostridium are also improved, and a large amount of ethanol clostridium is obtained for producing ethanol. Healthy strains are continuously and stably conveyed to a large anaerobic reactor, and the use cost of the freeze-dried powder is reduced.

Description

Technical Field

[0001] The present application relates to the technical field of ethanolic Clostridium culture, and in particular to a multi-stage gradient circulation culture method of ethanolic Clostridium. Background Art

[0002] The production of fuel ethanol using industrial tail gas fermentation is an emerging gas-liquid biofermentation technology. This technology uses the anaerobic microorganism Clostridium ethanolum to absorb CO in industrial tail gas, use CO gas as a carbon source, and convert inorganic carbon CO into organic carbon metabolites such as ethanol through its own metabolism.

[0003] At present, in order to maintain the stability and continuity of the industrial gas biofermentation process, it is necessary to regularly recover freeze-dried powder in medium-sized anaerobic reactors, cultivate healthy strains, and continuously replace healthy strains for large fermentation tanks. However, the preparation, storage, and transportation of freeze-dried powder strains may affect the success rate and duration of freeze-dried powder recovery. Once the freeze-dried powder strains are not recovered smoothly, it will affect the timeliness of inoculation of large fermentation tanks, and the healthy strains cannot be replaced in time for the fermentation tanks, which will affect the ethanol production. Summary of the invention

[0004] The present application provides a multi-stage gradient circulation culture method for ethanolic Clostridium to solve the following technical problem: how to ensure a stable supply of ethanolic Clostridium.

[0005] In a first aspect, the present invention provides a multi-stage gradient circulation culture method for ethanol Clostridium, the method being carried out under anaerobic and aseptic conditions, comprising the following steps:

[0006] Obtain ethanolic Clostridium glycerol bacteria;

[0007] resuscitating the ethanolic Clostridium glycerol bacteria to obtain an ethanolic Clostridium bacterial liquid;

[0008] inoculating the ethanolic Clostridium bacterial solution into a proliferation culture medium for proliferation culture;

[0009] The ethanol Clostridium ethanolate bacterial solution after proliferation culture is sequentially inoculated into a multi-stage reactor for multiple expansion culture and domestication to obtain domesticated ethanol Clostridium ethanolate;

[0010] using the domesticated ethanolic Clostridium to produce ethanol;

[0011] Wherein, the initial bacterial concentration of ethanol Clostridium in the multi-stage reactor is 0.15 g / L to 0.25 g / L.

[0012] Optionally, the method of sequentially inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a multi-stage reactor for multiple expansion culture and acclimation to obtain the acclimated ethanolic Clostridium specifically includes:

[0013] Inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a primary reactor for primary expansion culture and acclimatization to obtain a primary culture bacterial solution;

[0014] Repeat the above expansion and acclimatization steps n-1 times to obtain n-level culture solution;

[0015] When the n-level culture liquid meets the set conditions, the expansion and domestication are stopped to obtain the domesticated ethanol Clostridium;

[0016] The set conditions include at least one of the following: the total amount of ethanolic Clostridium in the n-1 level culture solution should ensure that the concentration of the n level culture solution is 0.15g / L to 0.25g / L; and the metabolism of ethanolic Clostridium is stable.

[0017] Optionally, the resuscitating the ethanolic Clostridium glycerol bacteria to obtain the ethanolic Clostridium bacterial liquid specifically includes: resuscitating the ethanolic Clostridium glycerol bacteria in an anaerobic bottle to obtain the ethanolic Clostridium bacterial liquid; the resuscitation ratio is 1 mL of glycerol bacteria resuscitated to 5 mL of culture medium.

[0018] Optionally, the types and concentrations of metal ions and vitamins in the proliferation medium and the multi-stage reactor are the same.

[0019] Optionally, the components of the proliferation medium include: peptone 14g / L~18g / L, yeast extract 8g / L~12g / L, fructose 8g / L~12g / L, sodium chloride 0.15g / L~0.25g / L, cysteine ​​hydrochloride 0.8g / L~1.2g / L, resazurin 0.4mL / L~0.6mL / L, and nutrient solution concentration 0.04mL / L~0.06mL / L.

[0020] Optionally, fructose is used as the carbon source in the proliferation medium.

[0021] Optionally, industrial tail gas with the same composition is used as a carbon source in the multi-stage reactor.

[0022] Optionally, the CO concentration in the industrial tail gas is 55% to 65%.

[0023] Optionally, the ventilation volume of the industrial tail gas during the multiple expansion and acclimatization is 0.02L / (min·g) to 0.1L / (min·g), that is, the ventilation volume required for each gram of bacteria is 0.02L / min to 0.1L / min.

[0024] Optionally, the method further comprises: preparing the acclimated ethanolic Clostridium that meets the preservation conditions into ethanolic Clostridium glycerol bacteria for standby use.

[0025] Optionally, the storage conditions specifically include: ethanol yield ≥ 30 g / L, survival time ≥ 90 days.

[0026] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0027] The embodiment of the present application provides a multi-stage gradient circulation culture method of ethanol Clostridium, which is carried out under anaerobic and aseptic conditions, and includes the following steps: obtaining ethanol Clostridium glycerol bacteria, using ethanol Clostridium glycerol bacteria as raw materials, and the recovery cost and success rate are higher than freeze-dried powder; resuscitating the ethanol Clostridium glycerol bacteria, with a high survival rate of recovery, to obtain ethanol Clostridium bacterial liquid, inoculating the ethanol Clostridium bacterial liquid into a proliferation culture medium for proliferation culture, so as to obtain a sufficient number of ethanol Clostridium for subsequent expansion and domestication. The ethanol Clostridium bacterial liquid after proliferation culture is sequentially inoculated into a multi-stage reactor for multiple expansion and domestication to obtain domesticated ethanol Clostridium; controlling the inoculation concentration of each reactor to be 0.15g / L to 0.25g / L, indicating that when the reactor is replaced, the ethanol Clostridium has reached the target bacterial concentration in the previous reactor, and the inoculation concentration can be guaranteed to remain unchanged when inoculated into the next reactor, thereby improving the expansion efficiency.

[0028] The method uses ethanol Clostridium glycerol as a raw material for a small amount of resuscitation, and gradually expands and domesticates the revived ethanol Clostridium in a sterile anaerobic environment, which not only controls the growth of miscellaneous bacteria from the source and has a high resuscitation rate, but also improves the production and metabolic capacity of ethanol Clostridium, obtains a large amount of ethanol Clostridium for ethanol production, realizes the continuous and stable delivery of healthy strains to large anaerobic reactors, and greatly reduces the cost of freeze-dried powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 A schematic diagram of a multi-stage gradient circulation culture method of ethanol Clostridium according to some embodiments of the present application;

[0032] Figure 2 Gram staining pictures of bacterial morphology in proliferation culture medium and various levels of anaerobic reactors

[0033] Figure 3 The monitoring results of CO gas utilization using the same nutrient element before and after the carbon source is switched in Example 1;

[0034] Figure 4The monitoring results of bacterial metabolism data using the same nutrient elements before and after the carbon source is switched in Example 1;

[0035] Figure 5 The monitoring results of CO gas utilization using different nutrient elements before and after the carbon source switching in Example 2;

[0036] Figure 6 The monitoring results of bacterial metabolism data using different nutrient elements before and after the carbon source switching in Example 2;

[0037] Figure 7 The figure is a schematic diagram of the principle of a multi-stage gradient circulation culture method of ethanolic Clostridium according to some embodiments of the present application. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0039] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0040] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions, refers to any combination of these items, including any combination of single or plural items; for example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, if the mass ratio of substance A, substance B and substance C is 1:2:3, then substance A, substance B and substance C should correspond to the proportional numbers in the proportional formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0042] Figure 1 A schematic diagram of a multi-stage gradient circulation culture method of ethanol Clostridium according to some embodiments of the present application;

[0043] like Figure 1 As shown, the present application embodiment provides a multi-stage gradient circulation culture method of ethanol Clostridium, which is carried out under anaerobic and aseptic conditions and includes the following steps:

[0044] S1, obtain ethanolic Clostridium glycerol bacteria;

[0045] S2, resuscitating the ethanolic Clostridium glycerol bacteria to obtain an ethanolic Clostridium bacterial liquid;

[0046] S3, inoculating the ethanolic Clostridium bacterial solution into a proliferation culture medium for proliferation culture;

[0047] S4, inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a multi-stage reactor in sequence for multiple expansion culture and domestication to obtain domesticated ethanolic Clostridium;

[0048] S5, using the domesticated ethanolic Clostridium to produce ethanol;

[0049] Wherein, the initial bacterial concentration of ethanol Clostridium in the multi-stage reactor is 0.15 g / L to 0.25 g / L.

[0050] In the above embodiment, ethanol Clostridium glycerol bacteria is a form of preservation of ethanol Clostridium, which is usually used for long-term preservation of strains. Using ethanol Clostridium glycerol bacteria as raw materials, the recovery cost and success rate are higher than freeze-dried powder; the method first uses a small amount of ethanol Clostridium glycerol bacteria for recovery, and the recovery survival rate is high, and then the recovered ethanol Clostridium bacterial liquid is proliferated and cultured to obtain a certain amount of ethanol Clostridium and then perform subsequent expansion and domestication; the ethanol Clostridium bacterial liquid after the proliferation culture is inoculated into a multi-stage reactor in turn for multiple expansion and domestication to obtain domesticated ethanol Clostridium; the initial cell concentration of ethanol Clostridium in each reactor is controlled to be 0.15g / L~0.25g / L. This concentration range indicates that when the reactor is replaced, the ethanol Clostridium has reached the target cell concentration in the previous reactor, and the inoculation concentration can be guaranteed to remain unchanged when inoculated into the next reactor, which helps to ensure the stable growth and reproduction of ethanol Clostridium in the reactor and improve the expansion efficiency. Exemplarily, the initial cell concentration of ethanol Clostridium in each stage of the reactor can be 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L, 0.19 g / L, 0.20 g / L, 0.21 g / L, 0.22 g / L, 0.23 g / L, 0.24 g / L and 0.25 g / L.

[0051] The multi-stage gradient circulation culture method of ethanolic Clostridium provided in the embodiment of the present application mainly solves the problem of how to ensure the supply of ethanolic Clostridium. Through the multi-stage gradient circulation culture method, efficient and stable culture of ethanolic Clostridium can be achieved, thereby meeting production needs. It has the following advantages:

[0052] Improve production efficiency: Multi-stage reactors can provide different environmental conditions to optimize the growth of ethanol Clostridium at different stages. Through multiple expansion and domestication, the growth rate and metabolic activity of ethanol Clostridium can be further improved, thereby improving production efficiency.

[0053] Guarantee product quality: The domesticated ethanol Clostridium has higher stability and adaptability, and can better adapt to changes in the production environment. This helps to ensure the quality and stability of products such as ethanol produced.

[0054] Reduce production costs: By optimizing culture conditions and improving production efficiency, production costs can be reduced. In addition, the multi-stage gradient circulation culture method can also achieve the recycling of ethanol Clostridium, further reducing production costs.

[0055] As an optional embodiment, the method of sequentially inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a multi-stage reactor for multiple expansion culture and domestication to obtain domesticated ethanolic Clostridium specifically includes:

[0056] Inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a primary reactor for primary expansion culture and acclimatization to obtain a primary culture bacterial solution;

[0057] Repeat the above expansion and acclimatization steps n-1 times to obtain n-level culture solution;

[0058] When the n-level culture liquid meets the set conditions, the expansion and domestication are stopped to obtain the domesticated ethanol Clostridium;

[0059] The set conditions include at least one of the following: the total weight of ethanol Clostridium in the n-1 level culture solution should ensure that the initial OD of the n-level ethanol Clostridium culture solution is 0.15g / L to 0.25g / L; and the ethanol yield of ethanol Clostridium is stable.

[0060] In the above embodiment, the ultimate goal of culturing through multi-stage reactors is to ensure that the total amount of ethanol Clostridium obtained finally reaches a certain amount to meet the needs of ethanol industrial production. For example, in the final stage of culture, the initial bacterial concentration is 0.15g / L~0.25g / L; or when the metabolic capacity of ethanol Clostridium is stable, such as the amount of strains, acetic acid metabolites, and ethanol metabolites all maintain an increasing trend, and the survival time and ethanol production can reach certain target values, the reactor replacement process can be stopped.

[0061] As an optional embodiment, the value of n is 3-5, that is, the cultured ethanolic Clostridium soln is expanded and domesticated for 3-5 times to obtain domesticated ethanolic Clostridium.

[0062] In the above embodiment, the more levels the reactor has, the more times it needs to be replaced. If the number of replacements exceeds a certain number, the bacterial solution may be contaminated, affecting the production of metabolites. If the reactor has fewer levels, the final total amount of ethanol Clostridium may not reach the target value.

[0063] As an optional embodiment, the method of sequentially inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a multi-stage reactor for multiple expansion culture and domestication to obtain domesticated ethanolic Clostridium specifically includes:

[0064] The ethanol Clostridium bacterial solution after proliferation culture is inoculated into a small reactor for primary expansion culture and acclimatization to obtain a primary culture solution;

[0065] Inoculating the secondary cultured bacterial solution into a medium-sized reactor for expansion and acclimatization to obtain a secondary cultured bacterial solution;

[0066] The secondary culture solution is inoculated into the culture medium of a large reactor for expansion and acclimatization to obtain a tertiary culture solution;

[0067] Repeat the above steps until the metabolism of Clostridium ethanolum is stabilized and the ethanol yield increases steadily.

[0068] In the above embodiment, the volume of the small reactor is 3L to 15L, and the volume of the medium-sized anaerobic reactor is 2m 3 ~20m 3 The volume of the large anaerobic reactor is 500m 3 ~1000m 3 .

[0069] As an optional embodiment, the resuscitating the ethanolic Clostridium glycerol bacteria to obtain the ethanolic Clostridium bacterial solution specifically includes: resuscitating the ethanolic Clostridium glycerol bacteria in an anaerobic bottle to obtain the ethanolic Clostridium bacterial solution; the resuscitation ratio is 1 ml of glycerol bacteria resuscitated to 5 ml of culture medium.

[0070] In the above embodiment, a 10 mL anaerobic bottle is used to resuscitate the ethanolic Clostridium glycerol bacteria, the resuscitation volume is small, the resuscitation conditions are easy to control, and the success rate is high.

[0071] As an optional embodiment, the types and concentrations of metal ions and vitamins in the proliferation medium and the multi-stage reactor are the same.

[0072] In the above embodiment, the reason for controlling the types and concentrations of metal ions and vitamins in the proliferation medium and the multi-stage reactor to be the same is that when the medium and the carbon source are replaced, the growth of the bacteria will not experience a stagnation period and ethanol Clostridium can grow smoothly.

[0073] As an optional embodiment, the components of the proliferation medium include: peptone 14g / L~18g / L, yeast extract 8g / L~12g / L, fructose 8g / L~12g / L, sodium chloride 0.15g / L~0.25g / L, cysteine ​​hydrochloride 0.8g / L~1.2g / L, resazurin 0.4mL / L~0.6mL / L, and nutrient solution concentration 0.04mL / L~0.06mL / L.

[0074] As an optional embodiment, fructose is used as the carbon source in the proliferation culture.

[0075] In the above embodiment, fructose is used as the carbon source in the proliferation culture because the operation is simple and the carbon source content is stable and controllable.

[0076] As an optional embodiment, the concentration of fructose is 8 g / L to 12 g / L. Exemplarily, the concentration of fructose may be 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L.

[0077] As an optional implementation, industrial tail gas with the same composition is used as a carbon source in the multiple expansion and acclimation.

[0078] In the above embodiment, the reason why industrial tail gas is used as the carbon source in the multiple expansion and domestication is that it helps to domesticate the metabolic ability of ethanol Clostridium to produce ethanol using industrial tail gas, and secondly, it saves energy and reduces industrial tail gas pollution.

[0079] As an optional implementation, in the industrial tail gas, the concentration of carbon monoxide is 55% to 65%, the concentration of nitrogen is 10% to 20%, the concentration of carbon dioxide is 20% to 30%, and the concentration of hydrogen is 2% to 5%.

[0080] In the above embodiment, the reason for controlling the CO concentration in the industrial tail gas to be 55% to 65% is that CO within this concentration range is more conducive to the metabolism of Clostridium ethanolicum.

[0081] As an optional embodiment, the gas components of the industrial tail gas are: CO concentration is 60%, CO 2 The concentration is 15%, N 2 The concentration is 20%, H 2 The concentration is 5%.

[0082] As an optional implementation, the ventilation volume of industrial tail gas during the multiple expansion and domestication follows the following principles: when the initial strain mass is about 2g to 5g, the initial bacterial concentration is 0.2g / L to 0.5g / L, and the initial ventilation volume is set to 0.1L / min to 0.2L / min, that is, the ventilation volume per gram of strain is 0.02L / min to 0.1L / min. As the strain increases, the ventilation volume is continuously increased to ensure that the ventilation volume per gram of strain is 0.02L / min to 0.1L / min to promote the growth of bacterial concentration.

[0083] In the above embodiment, controlling the ventilation volume of industrial tail gas is to control the carbon source concentration in the culture medium, and the appropriate carbon source concentration helps to improve the metabolic capacity of ethanol Clostridium and promote growth. Exemplarily, during the multiple expansion and acclimation, the ventilation volume of industrial tail gas required for each gram of ethanol Clostridium is 0.02L / min, 0.03L / min, 0.04L / min, 0.05L / min, 0.06L / min, 0.07L / min, 0.08L / min, 0.09L / min or 0.1L / min.

[0084] As an optional implementation, the method further comprises: preparing the ethanol Clostridium glycerol strain from the domesticated ethanol Clostridium that meets the preservation conditions for later use.

[0085] In the above embodiment, the acclimated ethanolic Clostridium that meets the preservation conditions is prepared as ethanolic Clostridium glycerol bacteria, so as to provide seed bacteria for the next recovery and realize the cyclic culture of ethanolic Clostridium.

[0086] As an optional implementation manner, the storage conditions specifically include: ethanol yield ≥ 30 g / L, survival time ≥ 90 days.

[0087] In the above embodiment, only when the above two conditions are met can it be proved that the ethanol Clostridium is of high quality and suitable for preservation. During the cultivation process, it is necessary to continuously detect the metabolic capacity of the ethanol Clostridium in each level of culture medium, and it is also necessary to perform Gram staining on the strains in each level of culture solution, and use a microscope to observe whether the solution is uniformly rod-shaped to avoid contamination by other forms of bacteria.

[0088] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are usually measured in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are measured in accordance with common international standards, conventional conditions or conditions recommended by the manufacturer.

[0089] Example 1

[0090] This embodiment provides a multi-stage gradient circulation culture method for ethanol Clostridium, which is carried out under anaerobic and aseptic conditions and comprises the following steps:

[0091] S1, obtaining ethanolic Clostridium glycerol bacteria, wherein the ethanolic Clostridium glycerol bacteria may be purchased or frozen;

[0092] S2, resuscitating the ethanolic Clostridium glycerol bacteria in a 10 mL anaerobic bottle, culturing at 37° C. for 1.5 days to obtain an ethanolic Clostridium bacterial solution; the resuscitation ratio is 1 mL of glycerol bacteria resuscitated to 5 mL of proliferation medium, and the components of the proliferation medium in the bottle are shown in Table 1;

[0093] S3, inoculating the ethanolic Clostridium bacterial solution into a 500 mL anaerobic bottle for further proliferation culture, with a volume of 300 mL, and culturing at 37° C. for 1 day to obtain the ethanolic Clostridium bacterial solution after proliferation culture, which is obviously turbid;

[0094] Table 1 Composition of proliferation medium in Example 1

[0095]

[0096] S4, inoculating the ethanolic Clostridium liquid after the proliferation culture into a small anaerobic reactor for expansion and acclimatization;

[0097] The volume of the small anaerobic reactor can be 3L-15L, the concentration of acetic acid is 1g / L, the concentration of cysteine ​​is 1g / L, the concentration of ammonium chloride is 0.5g / L, the concentration of nutrient solution (including iron ions, magnesium ions, calcium ions, potassium ions and vitamins) is 1.5x, the pH is about 5.0, the temperature is 37°C, and industrial tail gas (carbon monoxide concentration is 55% to 65%, nitrogen concentration is 10% to 20%, carbon dioxide concentration is 20% to 30%, hydrogen concentration is 2% to 5%) is used as the carbon source. When taking 15L reaction as an example, when the initial strain mass is about 2g, the initial bacterial concentration is 0.2g / L, and the initial ventilation volume is set to 0.1L / min to 0.15L / min. When the OD concentration reaches about 4g / L, the total strain mass of the single tank reactor is greater than 60g, and the primary culture solution is obtained;

[0098] S5. Inoculate the primary culture solution to 2m 3 -20m 3 The bacteria are expanded and domesticated in a medium-sized anaerobic reactor to obtain a secondary culture solution; the types and concentrations of metal ions and vitamins, temperature, pH and carbon source in the medium-sized anaerobic reactor are consistent with those in the small reactor;

[0099] S6, the secondary culture liquid is inoculated into a large anaerobic reactor for expansion and domestication. After testing, the initial bacterial concentration inoculated into the large anaerobic reactor is 0.25g / L, and by evaluating the metabolic data, the metabolic capacity of the ethanol Clostridium in this reactor is stable, and the reactor is no longer replaced. The ethanol Clostridium with strong adaptability and stable metabolic capacity in this reactor is further expanded, and the bacterial concentration is increased by adjusting the ventilation volume and the amount of culture liquid, and the ethanol yield of ethanol Clostridium is continuously improved. In addition, if the bacterial concentration is reduced, the secondary culture liquid can be continued from the upper reactor, that is, from the medium-sized reactor to ensure the bacterial concentration in the large reactor.

[0100] During the cultivation process, in each level of reactor, as the bacterial concentration increases, the corresponding amount of gas and chemical addition needs to be matched for bacterial reproduction and renewal;

[0101] In order to ensure the production of ethanol Clostridium, each stage reactor can have multiple reactors used in parallel, or the reactors at each stage can be connected in series to form multiple reactors of the same stage and operate simultaneously to increase the total weight of the inoculated bacteria concentration of the next stage reactor, ensure that the initial bacteria concentration of the next stage reactor is greater than 0.15-0.25g / L, and continuously domesticate and cultivate ethanol Clostridium liquid with a steadily increasing ethanol yield.

[0102] The metabolic capacity and morphology of ethanol Clostridium in medium-sized and large reactors are monitored, and ethanol Clostridium with ethanol production greater than 30g / L and continuous survival for more than 90 days are prepared into glycerol bacteria and frozen and stored in a -80°C refrigerator, so that the glycerol bacteria can be revived and expanded to continue to supply metabolically excellent strains to large reactors, thereby increasing the ethanol production of the production line.

[0103] In order to determine whether there are foreign bacteria in the bacterial solution of the anaerobic bottle and each level of reactor, Gram staining was also performed on the bacterial strains in each level of reactor while detecting the metabolic data. A microscope was used to preliminarily observe whether the morphology of ethanol Clostridium was uniform and rod-shaped. The results are as follows: Figure 2 As shown in the figure, it can be seen that no foreign bacteria are produced in each level of reactor, indicating that this method can avoid the generation of foreign bacteria and affect the growth and metabolism of ethanol Clostridium.

[0104] Example 2

[0105] This example provides a multi-stage gradient circulation culture method for ethanolic Clostridium, using the same steps as in Example 1, with the only difference being that the components in the proliferation culture medium are shown in Table 2.

[0106] Table 2 Proliferation medium components in Example 2

[0107]

[0108]

[0109] Table 3 Components of trace element solution

[0110] TSE solution components Component content nitrilotriacetic acid 2g / L <![CDATA[MnSO 4 ·H 2 O]]> 1g / L <![CDATA[Fe(SO 4 ) 2 (NH 4 ) 2 ·6H 2 O]]> 0.8g / L <![CDATA[CoCl 2 ·6H 2 O]]> 0.2g / L <![CDATA[ZnSO 4 ·7H 2 Oh]]> 0.2mg / L <![CDATA[CuCl 2 ·2H 2 Or]]> 0.02g / L <![CDATA[NaMoO 4 ·2H 2 Or]]> 0.02g / L <![CDATA[Na 2 This is 3 ]]> 0.02g / L <![CDATA[NiCl 2 ·6H 2 O]]> 0.02g / L <![CDATA[Na 2 WO 4 ·2H 2 O]]> 0.02g / L

[0111] Table 4 Composition of Wolfe's vitamin solution

[0112] Wolfe's vitamin solution ingredients Component content Biotin (Vitamin H) 2mg / L folic acid (vitamin B) 2mg / L Pyridoxine hydrochloride(Vitamin B6) 10mg / L Thiamine HCl (Vitamin B1) 5mg / L Riboflavin (Vitamin B2) 5mg / L Nicotinic acid (vitamin B3) 5mg / L calcium pantothenate (vitamin B5) 5mg / L Vitamin B12 0.1mg / L p-aminobenzoic acid (Vitamin Bx) 5mg / L Thioctic acid 5mg / L

[0113] The metabolic conditions of the ethanol Clostridium ethanolate solution after being transferred from the anaerobic bottle to the small anaerobic reactor in Example 1 and Example 2 were compared, and the results were as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 The metabolic status of the ethanolic Clostridium solution in Example 1 is shown in Figure 3It can be seen that since the nutrients in the anaerobic bottle and the reactor are the same, the bacteria are almost unaffected after switching the carbon source, and the bacterial metabolism increases significantly. The bacterial utilization rate reaches a plateau 2 to 3 days after the growth of the bacteria, but subsequently the ability of the bacteria to absorb CO2 gradually increases with the increase of the bacterial concentration.

[0114] Figure 4 The middle one is the metabolic data of ethanol Clostridium after the carbon source of ethanol Clostridium was changed from the anaerobic bottle to the anaerobic reactor in Example 2, accompanied by the change of nutrients. Figure 4 It can be seen that the alcoholic Clostridium consumed all the fructose in the anaerobic bottle within 17 hours, and the strain began to grow slowly. Until about the 5th day, the strain's ability to absorb CO gradually increased, and the strain's metabolism began to increase on the 7th day.

[0115] In summary, by changing the ion concentration (trace elements) and vitamin concentration in the proliferation medium to keep it consistent with that in the anaerobic reactor, that is, adding the same concentration of ions and vitamins when culturing with two carbon sources, the bacteria had almost no growth lag period after changing the carbon source. After moving from the anaerobic bottle to the reactor, it began to absorb CO, and the CO utilization rate gradually increased. Along with the decrease in fructose concentration in the culture medium, the bacterial metabolism increased significantly.

[0116] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0117] 1. The present application proposes a mode of using anaerobic bottles to revive glycerol bacteria. The survival rate of anaerobic bottles to revive glycerol bacteria is close to 100%, and different batches of ethanol Clostridium strains with different ethanol yields can be revived and stored at any time.

[0118] 2. Ethanol Clostridium has almost no lag period between the replacement of anaerobic bottle reactors and between each level of reactors, and its metabolic state is good, so it can continue to inoculate the next level of reactor.

[0119] 3. The whole process is carried out under sterile conditions to control the growth of bacteria from the source and create good conditions for the recovery of ethanol Clostridium.

[0120] 4. This culture mode prepares the strains with high alcohol production rate and vigorous metabolism into glycerol bacteria and preserves them, thus retaining excellent seed bacteria for subsequent replacement of healthy strains.

[0121] 5. Greatly reduce the cost of using freeze-dried powder and high-purity gas cylinders, thereby reducing production and operating costs.

[0122] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. A multi-stage gradient circulation culture method for ethanol Clostridium, the method being carried out under anaerobic and aseptic conditions, comprising the following steps: Obtain ethanolic Clostridium glycerol bacteria; resuscitating the ethanolic Clostridium glycerol bacteria to obtain an ethanolic Clostridium bacterial liquid; inoculating the ethanolic Clostridium bacterial solution into a proliferation culture medium for proliferation culture; The ethanol Clostridium ethanolate bacterial solution after proliferation culture is sequentially inoculated into a multi-stage reactor for multiple expansion culture and domestication to obtain domesticated ethanol Clostridium ethanolate; using the domesticated ethanolic Clostridium to produce ethanol; Wherein, the initial bacterial concentration of ethanol Clostridium in the multi-stage reactor is 0.15 g / L to 0.25 g / L.

2. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, wherein the ethanolic Clostridium liquid after proliferation culture is sequentially inoculated into a multi-stage reactor for multiple expansion culture and acclimation to obtain the acclimated ethanolic Clostridium specifically comprises: Inoculating the ethanolic Clostridium bacterial solution after proliferation culture into a primary reactor for primary expansion culture and acclimatization to obtain a primary culture bacterial solution; Repeat the above expansion and acclimatization steps n-1 times to obtain n-level culture solution; When the n-level culture liquid meets the set conditions, the expansion and domestication are stopped to obtain the domesticated ethanol Clostridium; The set conditions include at least one of the following: after the total amount of ethanol Clostridium in the n-1 level culture solution is transferred to the n level, the initial bacterial strain concentration OD of the n level culture solution is 0.15g / L to 0.25g / L; the metabolism of ethanol Clostridium is stable.

3. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, wherein the ethanolic Clostridium glycerol bacteria are revived to obtain the ethanolic Clostridium bacterial liquid specifically comprising: The ethanolic Clostridium glycerol bacteria were revived in an anaerobic bottle to obtain an ethanolic Clostridium bacterial solution; the revivification ratio was: 1 mL of glycerol bacteria was revived to 5 mL of culture medium.

4. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, wherein the types and concentrations of metal ions and vitamins in the proliferation culture medium and the multi-stage reactor are the same.

5. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, wherein fructose is used as the carbon source in the proliferation medium.

6. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, wherein industrial tail gas with the same composition is used as a carbon source in the multi-stage reactor.

7. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 6, wherein the CO concentration in the industrial tail gas is 55% to 65%.

8. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 7, wherein the ventilation rate of the industrial tail gas in the multi-stage reactor is 0.02 L / (min·g) to 0.1 L / (min·g).

9. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 1, further comprising: The acclimated ethanolic Clostridium that meets the preservation conditions is prepared as ethanolic Clostridium glycerol strain for later use.

10. The multi-stage gradient circulation culture method of ethanolic Clostridium according to claim 9, wherein the storage conditions specifically include: Ethanol productivity ≥30g / L, survival time ≥90 days.

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