JP2004208537A - Method for producing amino acid from methane-containing gas - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an amino acid from methane, by which the amino acid can efficiently be produced from the methane. <P>SOLUTION: This method for producing the amino acid from the methane is characterized by continuously or simultaneously performing (i) a process for producing methanol from the methane with a methane-assimilating bacterium and (ii) a process for producing the amino acid from the methanol obtained in the process (i) with a methanol-assimilating bacterium. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００７１】
ＯＤ２．０となるまで５０ｍｌの無機塩培地Ａで前培養したメタン資化細菌Ｍｅｔｈｙｌｏｃａｌｄｕｍ属Ｔ−０２５株（ＦＥＲＭ Ｐ−１８６４５）が入った培養槽Ａに、５０℃で、８０ｒｐｍの攪拌条件下で培養しながら、空気／メタン＝４：１混合気体を２５Ｌ／時間で培養液中へバブリング、並びに１０ｍｌ／時間で無機塩培地Ａの供給及び培養液の抜き取りを行った。定常状態時の培養槽Ａから抜き取られた培養液中のメタノール濃度を測定したところ、０．５重量％であった。培養槽Ａから抜き取られた培養液を遠心分離により菌体と上清に分離後、菌体は培養槽Ａ中へ戻し、培養上清は下記するように培養槽Ｂへ導入した。
【００７２】
ＯＤ３０となるまで５０ｍｌの無機塩培地Ａで前培養したＭｅｔｈｙｌｏｂａｃｉｌｌｕｓ属細菌（土壌から単離したメタノール資化細菌）が入った培養槽Ｂ中に、３０℃で、８０ｒｐｍの攪拌条件下で培養しながら、１０ｍｌ／時間で上記培養上清の供給及び培養液の抜き取りを行った。定常状態時の培養槽Ｂから抜き取られた培養液中には、２０ｍｇ／ｍｌのグルタミン酸が存在することが確認された。
【００７３】
以上の結果から、メタノールデヒドロゲナーゼ活性に対するメタンモノオキシゲナーゼ活性の比率が高いメタン資化細菌及びメタノール資化細菌を用いて、メタンからアミノ酸を効率的に生産できることが明らかとなった。
【００７４】
比較例１
４００ｍｌの培養槽Ｃを用いて、以下の試験を行った。
【００７５】
ＯＤ２．０となるまで１００ｍｌの無機塩培地Ａで前培養したメタン資化細菌ｍｅｔｈｙｌｏｃａｌｄｕｍ属Ｔ−０２５株（ＦＥＲＭ Ｐ−１８６４５）が入った培養槽Ｃ中に、５０℃で、８０ｒｐｍの攪拌条件下で培養しながら、空気／メタン＝４：１混合気体を２５Ｌ／時間で培養液中へバブリング、並びに１０ｍｌ／時間で無機塩培地Ａの供給・培養液の抜き取りを行った。
【００７６】
培養槽Ｃから抜き取られた培養液中のグルタミン酸濃度は、１ｍｇ／ｍｌと実施例１に比して顕著に低いことが確認された。この結果から、メタン資化細菌はメタノールを生産する能力に秀でているが、アミノ酸を生産することは困難であることが確認された。
【００７７】
【発明の効果】
本発明によれば、地球上に豊富に存在し、また量的にも価格的にも安定して供給される天然ガスを原料として、アミノ酸を低コストで生産することができる。
【図面の簡単な説明】
【図１】本発明のアミノ酸を生産する方法の実施態様（フロー）の一例を示す図である。図中、フロー１（上図）は工程（ｉ）及び（ｉｉ）を連続的に実施する態様の一例を、フロー２（下図）工程（ｉ）及び（ｉｉ）を同時に実施する態様の一例を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing an amino acid. More specifically, the present invention relates to a method for efficiently producing an amino acid from methane using a microorganism. Furthermore, the present invention relates to an apparatus for producing an amino acid.
[0002]
[Prior art]
In order for us to live a healthy daily life, it is necessary to take 20 kinds of amino acids in a well-balanced manner. However, in modern life, some amino acids are difficult to ingest, which tends to upset the nutritional balance. Today, supplementary foods (supplements) containing such deficient amino acids have become widespread and have become a means of supplementing deficient amino acids.
[0003]
On the other hand, amino acids other than glycine have optical isomers, and there are D-form and L-form, but most of naturally occurring amino acids including protein constituent units are L-amino acids, and amino acids used as foods are also L-amino acids. It is an L body. Since it is difficult to synthesize only L-amino acids that are useful as foods by a chemical synthesis method, amino acids produced by microorganisms are usually used as amino acids for foods.
[0004]
In the production of amino acids by such microorganisms, the cultivation of microorganisms usually uses an inexpensive carbon source such as molasses, in which case the supply amount, supply price is unstable, and transportation costs are high, There are problems such as generation of various wastes after use. Thus, studies have been made to synthesize amino acids using methanol as a readily available carbon source and methanol assimilating bacteria as microorganisms (for example, see Patent Document 1). However, this methanol has a problem that its price is high since it is produced by catalytic conversion by inputting a large amount of energy into methane.
[0005]
Therefore, if amino acids can be directly produced using methane supplied in large quantities as power generation or city gas, it is expected that amino acids can be produced at lower cost. However, methane-utilizing bacteria that grow using methane as a carbon source are generally difficult to culture, and so far, strains that produce large quantities of amino acids have not been bred. In addition, methane assimilating bacteria can oxidize methane to methanol, but since methane assimilating bacteria usually have higher methanol assimilating activity than methanol producing activity, the entire amount of produced methanol is oxidized to formaldehyde. There is also a problem that it cannot be recovered and used as methanol (for example, see Non-Patent Document 1).
[0006]
Against this background of the prior art, there has been a demand for the development of a technique for efficiently producing amino acids from methane that is abundant on the earth.
[0007]
[Patent Document 1]
JP-A-10-113196
[0008]
[Non-patent document 1]
Hanson et al., "Microbiological Reviews", U.S.A., June 1996, Vol. 60, No. 2, p. 439-471
[0009]
[Problems to be solved by the invention]
Then, an object of the present invention is to solve the above-mentioned conventional problems. More specifically, an object of the present invention is to provide a method for efficiently producing an amino acid from methane.
[0010]
[Means for Solving the Problems]
In order to achieve the above object, the present inventor has conducted repeated studies and found the following findings. That is,
Methane-utilizing bacteria, which are microorganisms that can use methane as a carbon source, usually oxidize methane to methanol and then immediately oxidize this methanol to formaldehyde, which is used as a cell component and energy source. Can not. However, certain methanotrophs secrete methanol into the growth medium because the rate of oxidizing methanol is slower than the rate of producing methanol.
Thus, amino acids can be stably produced from methane by supplying this methanol to methanol-utilizing bacteria.
[0011]
Based on the above findings, further studies have been made, and the present invention provides a method for producing the following amino acids.
Item 1. A method for producing an amino acid from methane, wherein the following steps (i) and (ii) are performed continuously or simultaneously:
(I) producing methanol from methane using methane-utilizing bacteria; and
(Ii) a step of producing an amino acid from methanol obtained in step (i) using a methanol-assimilating bacterium;
Item 2. Item 1. The method for producing an amino acid from methane according to Item 1, wherein the following steps (i) and (ii) are continuously performed:
(I) producing methanol from methane using methane-utilizing bacteria; and
(Ii) a step of producing an amino acid from methanol obtained in step (i) using a methanol-assimilating bacterium;
Item 3. Item 1. The method for producing an amino acid from methane according to Item 1 or 2, which comprises the following steps (1) to (5):
(1) a step of culturing methane-utilizing bacteria while supplying methane with aeration;
(2) a step of separating cells and a culture supernatant from the culture solution obtained in the above step (1);
(3) a step of culturing a methanol-assimilating bacterium using the culture supernatant obtained in the above step (2) as a medium,
(4) a step of separating cells and a culture supernatant from the culture solution obtained in the above step (3), and
(5) A step of collecting amino acids from the culture supernatant obtained in the above step (4) and reusing the generated waste liquid in the above step (1).
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the methane-utilizing bacterium is a methane-utilizing bacterium that grows at a temperature of 50 ° C or higher.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the methanotroph is a bacterium belonging to the genus Methylcardum.
Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the methane assimilating bacterium is a bacterium obtained under accession number FERM P-18645.
[0012]
Further, the present invention provides the following amino acid production apparatus.
Item 7. An amino acid producing apparatus comprising a methane-utilizing bacterium culturing tank and a methanol-utilizing bacterium culturing apparatus, or a mixed culturing tank of methane-utilizing bacterium and methanol-utilizing bacterium.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for producing an amino acid of the present invention comprises: (i) a step of producing methanol from methane using methane-assimilating bacterium; and (ii) an amino acid from methanol obtained in step (i) using methanol-assimilating bacterium. Is continuously or simultaneously performed.
[0014]
The methane assimilating bacterium used in the step (i) is not particularly limited as long as it can assimilate methane and accumulate methanol.
[0015]
As an example, the methane assimilating bacterium capable of accumulating methanol has, for example, a characteristic that the ratio of methane monooxygenase activity (hereinafter, referred to as MMO activity) to methanol dehydrogenase activity (hereinafter, referred to as MDH activity) is high. Methane assimilating bacteria.
[0016]
Here, the ratio of the MMO activity to the MDH activity can be measured, for example, by the following method. Methane-assimilating bacteria (30 ml of a 0.1 mg / ml concentration of the dry cell weight of the bacteria-containing medium) in a 70 ml closed vessel filled with a gas obtained by mixing methane and oxygen (or air) at a ratio of 1: 5. And shake for 30 minutes under the desired temperature conditions. Next, the amount (mol) of the reduced methane (hereinafter referred to as X) and the amount (mol) of methanol present in the medium (hereinafter referred to as Y) are measured. From the obtained measured values, the value of X / (XY) (hereinafter, the calculated value is referred to as the MMO / MDH ratio) is calculated.
[0017]
Examples of the methane assimilating bacteria used in the step (i) include those having an MMO / MDH ratio of more than 1, preferably 5 or more, and more preferably 10 or more. Further, the upper limit of the MMO / MDH ratio is not particularly limited.
[0018]
In addition, the methane-utilizing bacterium used in step (i) can produce methanol under specific conditions if it has a high methanol-producing ability under specific conditions. The production capacity may change reversibly.
In particular, a methane-assimilating bacterium that has a reversibly higher ratio of MMO activity to MDH activity under harsh conditions such as high temperature conditions, high pH, low pH, and high salt concentration as compared to mild conditions is preferably used. can do. Such methane assimilating bacteria can replenish the reducing power required for methanol production under mild conditions and perform efficient production of methanol under the above-mentioned severe conditions, so by repeating the severe and mild conditions This is advantageous in that methanol can be efficiently and continuously produced. The harsh condition here means, for example, as a high temperature condition, usually about 30 to 100 ° C., particularly about 50 to 70 ° C., more particularly about 60 to 65 ° C .; In particular, high pH conditions of about 8 to 9 or low pH conditions of usually about 4 to 7, especially about 5 to 6; and severe salt concentration conditions are usually about 1 to 100 g / l, especially 10 to 50 g / l. The following conditions can be mentioned. As an example of such a methane-utilizing bacterium, a methane-utilizing bacterium having an MMO / MDH ratio of more than 1 under such severe conditions, preferably 5 or more, more preferably 10 or more can be mentioned. . The term “mild conditions” as used herein refers to conditions suitable for the survival of the methane-assimilating bacterium, including the optimal conditions for survival. The optimal survival temperature is ± 10 ° C (preferably ± 5 ° C, more preferably ± 1 ° C). ), A pH within the optimal survival pH ± 2 (preferably ± 1, more preferably ± 0.2), and a salt concentration within the optimal survival salt concentration ± 10 g / l.
[0019]
Further, from the viewpoint of economy, as the methane assimilating bacterium used in the step (i), a thermophilic methane assimilating bacterium that can grow at about 50 ° C. or more is preferable. When cultivating methane-utilizing bacteria by supplying a culture medium and extracting a culture solution into a culture tank while aerating and supplying methane as a carbon source, use methane-utilizing bacteria that can grow at about 50 ° C. or higher. Thus, the amount of heat generated by the methane oxidation reaction is offset by the amount of heat required to maintain the culture temperature of the culture solution, so that efficient culture can be performed. In addition, by using such thermophilic methane assimilating bacteria, this high temperature culture solution can be introduced into the methanol assimilating bacteria culturing tank, which contributes to the mitigation of temperature drop due to natural heat dissipation in the methanol assimilating bacteria culturing tank. It is also advantageous in that it can be used.
[0020]
Such methane assimilating bacteria can be separated, for example, from a high temperature environment in which methane is present at a low concentration, such as a hot spring. In an environment where methane is present at a low concentration in this way, it is thought that there are methane assimilating bacteria whose MMO activity has been increased so that the methane at a low concentration can be used effectively. There is a high probability that methane-utilizing bacteria can be obtained.
[0021]
Further, the methane-utilizing bacteria that can be used in the present invention can also be obtained by subjecting a conventionally known methane-utilizing bacterium to a mutation treatment. For example, temperature-sensitive bacteria among methane assimilating bacteria are prepared by treating a conventionally known methane assimilating bacterium with a mutagen such as nitrosoguanidine, then applying the bacterial solution on a plate medium, and culturing at, for example, about 50 to 60 ° C. After forming a colony, a naphthalene solution is sprayed on the plate medium, the culture temperature is raised to, for example, about 60 to 65 ° C., and the culture is continued, and a colony whose surroundings turn red due to a temperature shift is selected. Can also be obtained. Examples of conventionally known methane assimilating bacteria include, for example, genus Methylocardum, genus Methylothermus, genus Methylomonas, genus Methylobacter thymosylus, genus Methylocystis, B) Bacteria belonging to the genus.
[0022]
A microorganism (Methylocardum sp. T-025) deposited as a methane assimilating bacterium that can be used in the present invention, preferably as a deposit number FERM P-18645 at the National Institute of Advanced Industrial Science and Technology. ) Can be exemplified.
[0023]
The methane assimilating bacterium may be one in which the bacterium is immobilized on various carriers used for immobilizing microorganisms, from the viewpoint of facilitating recovery and reuse of the bacterium.
[0024]
On the other hand, the methanol-assimilating bacterium used in the step (ii) is not particularly limited as long as it can grow using methanol as a carbon source. Methanol assimilating bacteria can produce amino acids from methanol. Examples of the methanol-assimilating bacteria that can be used in the present invention include, for example, the genus Bacillus, the genus Methylobacillus, the genus Methylomonas, the genus Pseudomonas, the genus Artobacterium, and the bacterium Methylobacterium ac. And bacteria belonging to the genus Methanomonas, the genus Methylophaga, the genus Methanolovorus, and the genus Methylophilus.
[0025]
The methanol-assimilating bacterium may be a wild strain isolated from the natural world such as soil, compost, lakes and marshes, but secretes specific amino acids from the viewpoint of easy recovery of the produced amino acids and productivity. Preferably, the bacterium has been bred to: Specifically, it is desirable that the bacterium be bred to secrete a specific amino acid at a concentration of about 5 times, more preferably about 30 times, in a culture solution. In particular, it is particularly preferable that the bacterium be bred so as to secrete a large amount of amino acids such as lysine, arginine, threonine, serine, fanylalanine, and glutamic acid, which usually have a low content in foods and easily cause insufficient intake. The methanol-assimilating bacterium may be one that mainly produces a single amino acid, or one that produces two or more amino acids. Such a methanol-assimilating bacterium can also be obtained from a strain distribution organization.
[0026]
As the methanol-assimilating bacterium, those in which the bacterium is immobilized on various carriers used for immobilizing microorganisms can be used from the viewpoint of facilitating recovery and reuse of the bacterium.
[0027]
The method for producing an amino acid of the present invention comprises a step of producing methanol from methane using the methane-assimilating bacterium (step (i)) and a step of producing methanol from the methanol obtained in step (i) using the methanol-assimilating bacterium. It has a step of producing an amino acid (step (ii)). In the present invention, the steps (i) and (ii) may be performed independently and continuously, or may be performed simultaneously in the same culture tank. Hereinafter, the steps (i) and (ii) will be described in detail separately for the case where they are performed independently and continuously (I) and the case where they are performed simultaneously (II).
[0028]
( I) Process (I) as well as (Ii) Is performed continuously
First, methanol is produced from methane using the methane assimilating bacteria (step (i)).
[0029]
The method for producing such methanol is not particularly limited. As an example, a method of adding a culture medium and the methane-utilizing bacteria to a culture tank and performing culture while supplying a mixed gas of methane and air (hereinafter, also referred to as a culture method) can be given.
[0030]
Here, as a culture tank for culturing methane-assimilating bacteria, an ordinary microorganism culture tank provided with a supply device or a supply mechanism for supplying a mixed gas of methane and air into a culture solution is exemplified.
[0031]
The medium used for cultivating methane-utilizing bacteria is nitrogen, phosphorus, sulfur, potassium, magnesium, a certain amount of copper ion, iron ion, and other trace amounts specific to the target methane-utilizing bacterium, which are usually required for the growth of microorganisms. An inorganic salt medium containing an element can be used, and either a synthetic culture solution or a natural culture solution may be used. In addition, since methane is supplied during culturing, the addition of other carbon sources is not essential. Further, as the main medium, wastewater generated by the practice of the present invention, that is, the culture supernatant after the final amino acid recovery can be reused.
[0032]
The methane concentration in the mixed gas supplied to the culture solution of the methane assimilating bacteria is usually about 1 to 80% by volume, preferably about 5 to 50% by volume, and more preferably about 20% by volume. The source of methane is not particularly limited, and examples thereof include methane produced from natural gas fields, methane in liquefied natural gas, methane in city gas, and methane obtained by methane fermentation of wastewater and waste. Can be used. The mixed gas may be a mixture of oxygen instead of air.
[0033]
The supply rate of the mixed gas is determined according to the size and depth of the culture tank, the mixing ratio of methane in the gas, the gas dispersion method, the culture temperature, the methane (and oxygen) utilization rate of the methane assimilating bacteria, and the like. What is necessary is just to set suitably. As an example, the supply rate of a mixed gas at which the supply gas amount per day is 0.1 to 10 L, preferably 0.2 to 5 L, more preferably 0.4 to 2 L per 1 L of the culture solution may be mentioned. it can.
[0034]
The method of supplying gas into the culture solution is not particularly limited, but from the viewpoint of improving the methane dissolution effect, aeration is performed from a deep position in the culture tank so as to form fine bubbles in the culture medium. It is preferable to vigorously agitate the inside.
[0035]
The methanol production rate of methane-assimilating bacteria is proportional to the dissolved methane concentration and the dissolved oxygen concentration, but if the dissolved oxygen concentration is too high, the methanol concentration is suppressed. Therefore, when the dissolved oxygen concentration in the culture solution is too high, by increasing the ratio of methane in the supplied mixed gas, reducing the supplied mixed gas amount, or performing operations such as gentle stirring. It is desirable to appropriately control the concentration of dissolved oxygen in the culture solution.
[0036]
Note that, in order to avoid a risk of explosion caused by mixing methane and air (or oxygen), methane and air (or oxygen) may be supplied alternately instead of the mixed gas.
[0037]
The culture temperature may be appropriately set according to the optimal growth temperature of the methane-assimilating bacterium to be used. In addition, other conditions such as the pH of the culture solution can be appropriately set according to the characteristics of the methane-utilizing bacteria used. For example, when using a methane-utilizing bacterium in which the ratio of MMO activity to MDH activity is reversibly increased under specific severe conditions compared to mild conditions, the culture conditions may be changed to the specific severe conditions. The conditions are set so that methanol is produced efficiently, and when the rate of methanol accumulation in the culture medium decreases, the conditions are returned to mild conditions and the replenishing power required for methanol production is replenished. Efficient production of methanol becomes possible.
[0038]
The culture of methane-utilizing bacteria can be carried out, for example, by continuously or intermittently supplying a culture medium and extracting a culture solution into a culture tank. In this case, the method of supplying the culture medium and extracting the culture medium is not particularly limited. For example, a method of successively extracting the culture medium and supplying the culture medium while performing the culture, or extracting the culture liquid and supplying the culture medium while performing the culture. At the same time. The residence time in such a culture method differs depending on the type of bacteria used, the supply rate of methane, the size of the culture tank, the culture form, and the like, and cannot be uniformly defined. Days, preferably about 0.5 to 5 days, and more preferably about 1 to 3 days.
[0039]
Further, the culture of methane assimilating bacteria can also be performed by, for example, culturing while supplying a mixed gas without supplying a new medium and extracting a culture solution. The culturing time in such a case varies depending on the type of bacteria used, the supply rate of methane, and the like, and cannot be uniformly defined, but as an example, 0.5 to 10 days, preferably 0.5 to 5 days. Days, more preferably about 1 to 3 days.
[0040]
The culture solution thus obtained contains methanol produced by methane assimilating bacteria, and the culture solution is separated from the bacterial cells and the culture supernatant using a solid-liquid separation means such as centrifugation or membrane separation. (Hereinafter, referred to as a methanol-containing culture supernatant), the methanol-containing culture supernatant is subjected to the treatment in step (ii) described below. The separated cells can be returned to the culture solution of the methane-utilizing bacterium culturing tank, if necessary, and used for the production of methanol. The separated cells may be reused as a seed for producing methanol in the step (i) or may be effectively used as feed.
[0041]
In addition, as an example of a method for producing methanol from methane using methane-utilizing bacteria, a method in which methane-utilizing bacteria are held on a carrier and a mixed gas of methane and air is brought into contact with the carrier in a gas phase (hereinafter, referred to as gas). Phase contact method).
[0042]
The carrier for retaining the methane assimilating bacteria is not particularly limited. And a carrier having the formula:
[0043]
The amount of methane assimilating bacteria to be retained in the carrier depends on the type of carrier to be used, the type of methane assimilating bacterium, and cannot be uniformly defined. The ratio can be about 1 to 10000 parts by weight, preferably about 1 to 1000 parts by weight.
[0044]
Further, the carrier holding the methane-utilizing bacteria, if necessary, the growth of the bacteria such as carbon, nitrogen, phosphorus, sulfur, potassium, magnesium, calcium, iron, etc. required for the growth of the methane-utilizing bacteria May be included. Further, the carrier holding the methane assimilating bacteria preferably has a water content of, for example, about 50% by weight or less, preferably about 40% by weight or less. The lower limit of the water content may be any range as long as the methane assimilating bacteria can survive, and may be, for example, 30% by weight.
[0045]
The methane concentration in the mixed gas to be contacted is not limited, but may be, for example, about 5 to 90% by volume, preferably about 10 to 50% by volume. Further, oxygen may be used instead of air as the mixed gas.
[0046]
The temperature of the atmosphere when the mixed gas is brought into contact with the carrier holding the methane assimilating bacteria may be appropriately set according to the growth temperature of the methane assimilating bacteria to be used.
[0047]
The method for bringing the mixed gas into contact with a carrier holding methane assimilating bacteria is not particularly limited. For example, a method in which a carrier holding methane-utilizing bacteria and the above-mentioned mixed gas coexist in a closed space, a method in which the above-mentioned mixed gas is passed through a carrier holding methane-utilizing bacteria, and the like can be mentioned.
[0048]
Ethanol can be obtained in a gaseous or liquid state by such a gas phase contact method, and the ethanol is subjected to the process of step (ii) described below.
[0049]
Next, an amino acid is produced from the methanol obtained in the step (i) using the methanol-assimilating bacterium (step (ii)).
[0050]
When the step (i) is carried out by a culture method, the production of such amino acids can be carried out by culturing the methanol-assimilating bacterium using the above-mentioned methanol-containing culture supernatant as a medium.
[0051]
In the cultivation of the methanol-assimilating bacterium, the culture supernatant containing methanol may be used as it is as a medium, but if necessary, nutrient components and the like may be added or the pH may be adjusted.
[0052]
As a culture device for culturing methanol-assimilating bacteria, a culture device similar to that used in the culture method of the above-mentioned step (i) can be used except that a device for supplying methane is not essential.
[0053]
The cultivation of the methanol-assimilating bacterium is preferably performed by aerating air or oxygen and stirring. The culture temperature, the pH of the culture solution, the concentration of dissolved oxygen in the culture solution, and the like may be appropriately set according to the characteristics of the methanol-assimilating bacterium used.
[0054]
The cultivation of the methanol-assimilating bacterium can be performed, for example, by continuously or intermittently supplying a culture medium and extracting a culture solution into a culture tank. In this case, the method of supplying the culture medium and extracting the culture medium is not particularly limited. For example, a method of successively extracting the culture medium and supplying the culture medium while performing the culture, or extracting the culture liquid and supplying the culture medium while performing the culture. At the same time. The residence time in such a culture method varies depending on the type of bacteria used, the concentration of methanol in the medium, the size of the culture tank, the culture form, etc., and cannot be uniformly defined. Days, preferably about 0.5 to 5 days, and more preferably about 1 to 3 days.
[0055]
Furthermore, the cultivation of the methanol-assimilating bacterium can be performed, for example, by batch or fed-batch culture. The culturing time in such a case depends on the type of bacteria used, the concentration of methanol in the medium, and the like, and cannot be uniformly defined, but as an example, 0.5 to 10 days, preferably 0.5 to 5 days. Days, more preferably about 1 to 3 days.
[0056]
In the production of amino acids in step (ii), when step (i) is carried out by a gas phase contact method, for example, a methanol-assimilating bacterium using ethanol obtained in step (i) as a carbon source is used. Is prepared by culturing a methanol-assimilating bacterium under the same conditions as those described above using a culture medium capable of growing. Furthermore, when the step (i) is carried out by a gas phase contact method, the production of the amino acid may be carried out, for example, when methanol is obtained as a gas, for example, nitrogen, phosphorus, sulfur, which are usually required for the growth of microorganisms. Prepare an inorganic salt medium containing potassium, magnesium, a certain amount of copper ion, iron ion, and other trace elements specific to the target methanol-assimilating bacterium, and use this to aeration-supply the methanol-containing gas (for example, by bubbling). Except for the supply, the culture can be carried out by culturing a methanol-assimilating bacterium under the same conditions as those described above.
[0057]
As described above, performing the steps (i) and (ii) in separate and independent culture tanks involves separately culturing each of the methane assimilating bacteria and the methanol assimilating bacterium according to the optimal conditions of each bacterium. This is advantageous in that control is possible.
[0058]
( II) Process (I) as well as (Ii) In the same culture tank
Using the methane assimilating bacteria and methanol assimilating bacteria, methanol is produced from methane in the same culture tank, and amino acids are produced from the methanol (steps (i) and (ii)). The production of such amino acids is carried out by adding a medium, the methane-utilizing bacterium and the methanol-utilizing bacterium to a culture tank, supplying a mixed gas of methane and air, and performing a mixed culture of the both bacteria.
[0059]
The methane assimilating bacterium and the methanol assimilating bacterium used here may be those that have the same growth temperature and pH range for both bacteria. Further, for example, when a methane assimilating bacterium is used which has a reversible increase in the ratio of MMO activity to MDH activity as compared with mild conditions under specific severe conditions, it may be used as a methanol assimilating bacterium. It is desirable to select bacteria that can grow under such specific harsh conditions.
[0060]
In such a mixed culture, the culture tank, the culture medium, the mixed gas of methane and air, and the method of supplying the mixed gas are described in the step (i) in the case where the above-described steps (i) and (ii) are performed in an independent culture tank. Similar to the ones and methods used. The culture temperature and the pH in the culture solution can be appropriately set according to the characteristics of both bacteria used. Further, for example, when a bacterium in which the ratio of MMO activity to MDH activity is reversibly higher than that under mild conditions under specific severe conditions is used as a methane-assimilating bacterium, the cultivation conditions are also adjusted to the specific severe conditions. Under such conditions, the productivity of methanol is improved, and more efficient production of amino acids becomes possible.
[0061]
Such mixed culture can be carried out, for example, by continuously or intermittently supplying a culture medium into a culture tank and extracting a culture solution. In this case, the method of supplying the culture medium and extracting the culture medium is not particularly limited. For example, a method of successively extracting the culture medium and supplying the culture medium while performing the culture, or extracting the culture liquid and supplying the culture medium while performing the culture. At the same time. The residence time in such a culture method differs depending on the type of both bacteria used, the supply rate of methane, the size of the culture tank, the culture form and the like, and cannot be uniformly defined. 10 days, preferably about 0.5 to 5 days, more preferably about 1 to 3 days.
[0062]
In addition, such mixed culture can be performed, for example, by culturing while supplying a mixed gas without supplying a new medium and extracting a culture solution.
The cultivation time in such a case depends on the type of both bacteria used, the supply rate of methane and the like, and cannot be uniformly defined, but as an example, 0.5 to 10 days, preferably 0.5 to 10 days. 5 days, more preferably about 1 to 3 days.
[0063]
As described above, the culture solution obtained by performing the steps (i) and (ii) independently or in the same culture tank contains an amino acid produced by a methanol-assimilating bacterium.
The culture solution is separated into bacterial cells and amino acid-containing culture supernatant by solid-liquid separation means such as centrifugation or membrane separation. The amino acid-containing culture supernatant thus obtained can be used as an amino acid for feed by directly subjecting it to a drying treatment, and if necessary, an adsorption separation treatment with an ion-exchange resin or the like, or other commonly used in the art. By subjecting it to a certain purification treatment, it can be converted to an amino acid for feed or food.
[0064]
The wastewater generated by the amino acid purification treatment can be reused as one component of a culture medium for culturing methane-utilizing bacteria. In such reuse, for example, nitrogen, phosphorus, sulfur, potassium, magnesium, copper ions, iron ions, and other appropriate amounts of trace elements specific to the target methane assimilating bacterium are added to the wastewater to be used as a medium. be able to. Cells isolated by solid-liquid separation can be used as inoculants of methanol-utilizing bacteria or a mixed bacterium of methane-utilizing bacteria and methanol-utilizing bacteria. Can also.
[0065]
As a preferred embodiment of the method for producing an amino acid from methane according to the present invention, a method comprising the following steps (1) to (5) can be mentioned:
(1) a step of culturing methane-utilizing bacteria while supplying methane with aeration;
(2) a step of separating cells and a culture supernatant from the culture solution obtained in the above step (1);
(3) a step of culturing a methanol-assimilating bacterium using the culture supernatant obtained in the above step (2) as a medium,
(4) a step of separating cells and a culture supernatant from the culture solution obtained in the above step (3), and
(5) A step of collecting amino acids from the culture supernatant obtained in the above step (4) and reusing the generated waste liquid in the above step (1).
[0066]
Further, as an example of a preferred embodiment of the present invention, a flow chart of a method of performing steps (i) and (ii) independently and continuously and a method of performing steps (i) and (ii) simultaneously are shown in the respective flow charts. Is shown in FIG.
[0067]
Further, as described above, the step (i) of producing methanol from methane using methane-assimilating bacteria and the step (ii) of producing amino acids from methanol obtained in step (i) using methanol-assimilating bacteria ) Can be performed continuously or simultaneously to produce amino acids from methane. That is, amino acids can be produced by using the methane-utilizing bacterium culturing tank and the methanol-utilizing bacterium culturing apparatus described above or the mixed culturing tank of methane-utilizing bacterium and methanol-utilizing bacterium. Therefore, the present invention is further an apparatus for producing an amino acid comprising a methane-utilizing bacterium culturing tank and a methanol-utilizing bacterium culturing apparatus, or a mixed culturing tank of methane-utilizing bacterium and a methanol-utilizing bacterium. The apparatus for producing an amino acid of the present invention includes a device for solid-liquid separation of a methane-assimilating bacterium culture solution, and a solid-liquid separation of a methanol-assimilating bacterium culture solution or a mixed culture solution of methane-assimilating bacteria and tanol-assimilating bacteria. , An amino acid recovery device, and the like.
[0068]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited at all by the examples and the like.
[0069]
Example 1
The following test was performed using a 200 ml culture tank A for culturing methane assimilating bacteria and a 200 ml culture tank B for culturing methanol assimilating bacteria. As the inorganic salt medium A, a medium having the composition shown in Table 1 below was used.
[0070]
[Table 1]

[0071]
Under agitation conditions at 50 ° C. and 80 rpm in a culture tank A containing a methane assimilating bacterium, Methylocardum sp. While culturing, a mixed gas of air / methane = 4: 1 was bubbled into the culture at 25 L / hour, and the inorganic salt medium A was supplied and the culture was withdrawn at 10 ml / hour. The concentration of methanol in the culture solution extracted from the culture tank A in the steady state was measured and was 0.5% by weight. After the culture solution withdrawn from the culture tank A was separated into cells and supernatant by centrifugation, the cells were returned to the culture tank A, and the culture supernatant was introduced into the culture tank B as described below.
[0072]
In a culture tank B containing a bacterium belonging to the genus Methylobacillus (a methanol-assimilating bacterium isolated from soil) pre-cultured in 50 ml of an inorganic salt medium A until the OD reaches 30, the cells are cultured at 30 ° C. under stirring conditions of 80 rpm. The culture supernatant was supplied and the culture solution was withdrawn at 10 ml / hour. It was confirmed that 20 mg / ml of glutamic acid was present in the culture solution extracted from the culture tank B in the steady state.
[0073]
From the above results, it has been clarified that an amino acid can be efficiently produced from methane using methane-utilizing bacteria and methanol-utilizing bacteria having a high ratio of methane monooxygenase activity to methanol dehydrogenase activity.
[0074]
Comparative Example 1
The following test was performed using the culture tank C of 400 ml.
[0075]
In a culture tank C containing a methane-utilizing bacterium methylocardum genus T-025 strain (FERM P-18645) precultured in 100 ml of an inorganic salt medium A until the OD becomes 2.0, stirring conditions of 50 rpm and 80 rpm are set. While culturing, the air / methane = 4: 1 mixed gas was bubbled into the culture at 25 L / hour, and the inorganic salt medium A was supplied and the culture was withdrawn at 10 ml / hour.
[0076]
It was confirmed that the concentration of glutamic acid in the culture solution extracted from the culture tank C was 1 mg / ml, which was significantly lower than that in Example 1. From these results, it was confirmed that methane assimilating bacteria have excellent ability to produce methanol, but it is difficult to produce amino acids.
[0077]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, amino acids can be produced at low cost using natural gas, which is abundant on the earth and supplied stably in quantity and price, as a raw material.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of an embodiment (flow) of a method for producing an amino acid of the present invention. In the figure, a flow 1 (upper figure) is an example of an embodiment in which steps (i) and (ii) are continuously performed, and a flow 2 (lower figure) is an example of an embodiment in which steps (i) and (ii) are simultaneously performed. Show.

Claims (7)

A method for producing an amino acid from methane, wherein the following steps (i) and (ii) are performed continuously or simultaneously:
(I) a step of producing methanol from methane using methane-utilizing bacteria; and (ii) a step of producing amino acids from methanol obtained in step (i) using methanol-utilizing bacteria. The method for producing an amino acid from methane according to claim 1, wherein the following steps (i) and (ii) are continuously performed:
(I) a step of producing methanol from methane using methane-utilizing bacteria; and (ii) a step of producing amino acids from methanol obtained in step (i) using methanol-utilizing bacteria. The method for producing an amino acid from methane according to claim 1 or 2, comprising the following steps (1) to (5):
(1) a step of culturing methane-utilizing bacteria while supplying methane with aeration;
(2) a step of separating cells and a culture supernatant from the culture solution obtained in the above step (1);
(3) a step of culturing a methanol-assimilating bacterium using the culture supernatant obtained in the above step (2) as a medium,
(4) a step of separating bacterial cells and a culture supernatant from the culture solution obtained in the above step (3), and (5) an amino acid is recovered from the culture supernatant obtained in the above step (4) to produce A step of reusing the waste liquid in the above step (1). The method according to any one of claims 1 to 3, wherein the methane-utilizing bacterium is a methane-utilizing bacterium that grows at a temperature of 50 ° C or higher. The method according to any one of claims 1 to 4, wherein the methane assimilating bacterium is a bacterium belonging to the genus Methylcardum. The method according to any one of claims 1 to 5, wherein the methane assimilating bacterium is a bacterium obtained under accession number FERM P-18645. An amino acid producing apparatus comprising a methane-utilizing bacterium culturing tank and a methanol-utilizing bacterium culturing apparatus, or a mixed culturing tank of methane-utilizing bacterium and methanol-utilizing bacterium.

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