JPH11253153A - Cultivation of ammonia oxidative bacterium and measurement of number of bacteria - Google Patents
Cultivation of ammonia oxidative bacterium and measurement of number of bacteriaInfo
- Publication number
- JPH11253153A JPH11253153A JP8296098A JP8296098A JPH11253153A JP H11253153 A JPH11253153 A JP H11253153A JP 8296098 A JP8296098 A JP 8296098A JP 8296098 A JP8296098 A JP 8296098A JP H11253153 A JPH11253153 A JP H11253153A
- Authority
- JP
- Japan
- Prior art keywords
- ammonia
- bacteria
- heterotrophic
- oxidizing bacteria
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、アンモニア酸化細
菌の培養法及び菌数計測方法に関する。The present invention relates to a method for culturing ammonia-oxidizing bacteria and a method for counting the number of bacteria.
【０００２】[0002]
【従来の技術】アンモニア酸化細菌は、独立栄養細菌で
あり、通常アンモニアを含む無機培地中で純粋培養され
る。しかし、その増殖速度は遅く、基質濃度、生産物
（亜硝酸、硝酸）濃度、溶存酸素濃度などの影響を受け
やすいため、高濃度に培養することが困難であった。ま
た、純粋培養を試みた場合、培養中の生理活性の発現は
しばしば不安定であり、一定の菌体濃度に到達するまで
に予想外の時間を必要とすることも希ではない。また、
アンモニア酸化細菌には、高濃度のアンモニアの存在で
高活性を発揮するものと、高濃度アンモニアによって増
殖が阻害を受け、低濃度のアンモニアの存在で高活性を
発揮するものがあることが既に確認されている（特開平
９−４７７８７号公報参照）。これらのうち特に、高濃
度アンモニアによって増殖が阻害を受け、低濃度アンモ
ニアの存在で高活性を発揮する基質感受性（以下、低濃
度アンモニア適応種と略記することがある）のアンモニ
ア酸化細菌は、特に増殖速度が極端に遅く、最確値（Ｍ
ＰＮ）法により低濃度アンモニア適応種のアンモニア酸
化細菌の菌数を計測するには、４週間から８週間の培養
期間を必要とする上、現在充分に確立した継代培養法が
なく、植え継ぎを繰り返すうちに死滅してしまうことが
多いという問題があった。2. Description of the Related Art Ammonia-oxidizing bacteria are autotrophic bacteria and are usually purely cultured in an inorganic medium containing ammonia. However, its growth rate is slow, and it is difficult to culture at a high concentration because it is easily affected by substrate concentration, product (nitrite, nitrate) concentration, dissolved oxygen concentration and the like. In addition, when a pure culture is attempted, the expression of the physiological activity during the culture is often unstable, and it is not rare that an unexpected time is required to reach a certain cell concentration. Also,
It has already been confirmed that some ammonia-oxidizing bacteria exhibit high activity in the presence of high concentration of ammonia, while others exhibit high activity in the presence of low concentration of ammonia because growth is inhibited by high concentration of ammonia (See Japanese Patent Application Laid-Open No. 9-47787). Among these, especially substrate-sensitive (hereinafter sometimes abbreviated as low-concentration ammonia-adaptive species) ammonia-oxidizing bacteria whose growth is inhibited by high-concentration ammonia and exhibit high activity in the presence of low-concentration ammonia, The growth rate is extremely slow and the most probable value (M
In order to measure the number of ammonia-oxidizing bacteria of low-concentration ammonia-adapted species by the PN) method, a culture period of 4 to 8 weeks is required. There was a problem that it often died during the repetition of.
【０００３】[0003]
【発明が解決しようとする課題】本発明は、低濃度アン
モニア適応種のアンモニア酸化細菌の増殖速度の向上を
図り、また、安定した増殖を可能にし、さらに培養の誘
導期を短縮することができ、培養期間を短縮した効率の
よい培養法及びＭＰＮ法による菌数計測に必要な培養期
間を短縮した菌数計測方法を提供することを目的とす
る。SUMMARY OF THE INVENTION The present invention can improve the growth rate of low-concentration ammonia-adapted species of ammonia-oxidizing bacteria, enable stable growth, and shorten the induction period of culture. It is another object of the present invention to provide an efficient culture method in which the culture period is shortened, and a method for counting the number of bacteria necessary for counting the number of bacteria by the MPN method.
【０００４】[0004]
【課題を解決するための手段】本発明は、アンモニア酸
化細菌の培養又は培養を伴う菌数測定の際に、従来は混
入を避けるために様々な努力がなされてきた従属栄養細
菌を故意に混入させるか、又はその従属栄養細菌の培養
ろ液を添加することによって上記の目的を達成したもの
である。DISCLOSURE OF THE INVENTION The present invention is directed to the heterogeneous vegetative bacterium which has been deliberately mixed with various attempts to avoid contamination when culturing or counting the number of ammonia-oxidizing bacteria. Or the addition of a culture filtrate of the heterotrophic bacterium.
【０００５】すなわち、本発明は、高濃度アンモニアに
よって増殖が阻害を受け、低濃度アンモニアに親和性の
高い、基質感受性のアンモニア酸化細菌を培養するた
め、該アンモニア酸化細菌の培養液中に従属栄養細菌、
その培養液又は培養ろ液を添加することを特徴とするア
ンモニア酸化細菌の培養法を提供するものである。本発
明はさらに、高濃度アンモニアによって増殖が阻害を受
け、低濃度アンモニアに親和性の高い、基質感受性のア
ンモニア酸化細菌の菌数計測を最確値法によって行うた
め、該アンモニア酸化細菌の培養液中に従属栄養細菌、
その培養液又は培養ろ液を添加することを特徴とするア
ンモニア酸化細菌の菌数計測方法を提供する。That is, the present invention provides a method for cultivating substrate-sensitive ammonia-oxidizing bacteria whose growth is inhibited by high-concentration ammonia and has high affinity for low-concentration ammonia, and Bacteria,
It is intended to provide a method for culturing ammonia-oxidizing bacteria, which comprises adding the culture solution or the culture filtrate. The present invention furthermore, growth is inhibited by high-concentration ammonia, high affinity for low-concentration ammonia, the number of substrate-sensitive ammonia-oxidizing bacteria to be performed by the most probable method, the culture of the ammonia-oxidizing bacteria Heterotrophic bacteria,
A method for counting the number of ammonia-oxidizing bacteria, characterized by adding the culture solution or the culture filtrate.
【０００６】[0006]
【発明の実施の形態】本発明は、高濃度（１０００ｍｇ
−Ｎ／Ｌ以上）アンモニアによって増殖が阻害を受け、
低濃度アンモニアに親和性の高い、基質感受性のアンモ
ニア酸化細菌、すなわち、低濃度アンモニア適応種のア
ンモニア酸化細菌を培養する方法及びその培養を伴う菌
数計測方法を対象とするものである。本発明の菌数計測
方法においても、本発明の培養法と同一の培養を行うの
で、以下、培養法と菌数計測方法とを区別せずに説明す
る。また、本発明の方法は、回分法でも連続的培養にも
適用することができる。BEST MODE FOR CARRYING OUT THE INVENTION The present invention has a high concentration (1000 mg).
-N / L or more) The growth is inhibited by ammonia,
The present invention is directed to a method of cultivating substrate-sensitive ammonia-oxidizing bacteria having a high affinity for low-concentration ammonia, that is, a method of culturing ammonia-oxidizing bacteria of low-concentration ammonia-adaptive species and a method of counting the number of bacteria accompanying the culturing. In the method for counting the number of bacteria of the present invention, the same culture as that of the culture method of the present invention is performed. Therefore, the following description will be made without distinguishing between the culture method and the method for counting the number of bacteria. Further, the method of the present invention can be applied to a batch method or a continuous culture.
【０００７】本発明において、低濃度アンモニア適応種
のアンモニア酸化細菌とは、１０００ｍｇ−Ｎ／Ｌ以上
の濃度のアンモニア性窒素を含む培地で増殖に阻害を受
け、かつ、１〜５０ｍｇ−Ｎ／Ｌの濃度のアンモニア性
窒素を含む培地でよく増殖する菌種をいう。また、培地
としては、上記のアンモニア濃度範囲を有する無機培地
であれば、特に制限はなく、菌株の保存用培地、特に継
代培養用液体培地を使用するのが好ましい。In the present invention, a low-concentration ammonia-adaptive species of ammonia-oxidizing bacterium is inhibited from growing in a medium containing a concentration of at least 1000 mg-N / L of ammonia nitrogen, and is 1 to 50 mg-N / L. Refers to a bacterial species that grows well in a medium containing ammonia nitrogen at a concentration of The medium is not particularly limited as long as it is an inorganic medium having the above-mentioned ammonia concentration range, and it is preferable to use a medium for storing strains, particularly a liquid medium for subculture.
【０００８】上記のように、本発明においては、低濃度
アンモニア適応種のアンモニア酸化細菌の培養液に従属
栄養細菌、その培養液（菌体懸濁液）又は培養ろ液を添
加する。ここで、アンモニア酸化細菌の培養液とは、ア
ンモニア酸化細菌の菌体懸濁液を意味する。また、アン
モニア酸化細菌の培養液（菌体懸濁液）に従属栄養細
菌、その培養液又は培養ろ液を添加する場合、これらを
予めアンモニア酸化細菌培養用の新しい培地に加えてか
ら、アンモニア酸化細菌の菌体懸濁液に添加することも
できる。As described above, in the present invention, a heterotrophic bacterium, a culture solution thereof (cell suspension) or a culture filtrate is added to a culture solution of a low-concentration ammonia-adaptive species of ammonia-oxidizing bacteria. Here, the culture solution of ammonia oxidizing bacteria means a cell suspension of ammonia oxidizing bacteria. When a heterotrophic bacterium, a culture solution thereof or a culture filtrate thereof is added to a culture solution (cell suspension) of ammonia-oxidizing bacteria, these are added to a new medium for culturing ammonia-oxidizing bacteria beforehand. It can also be added to a bacterial cell suspension.
【０００９】ここで、従属栄養細菌としては、普通寒天
培地又は Tripticate Soy Broth （ＴＳＢ）培地又はこ
れらを希釈した培地に生育することが可能な従属栄養細
菌を用いることができ、例えば、パラコッカス（Paraco
ccus）属、シュウドモナス（Pseudomonas ）属、フラボ
バクテリウム（Flavobacterium）属、アシネトバクター
（Acinetobacter ）属、バルクホルデリア（Burkholder
ia）属、エシェリヒア（Escherichia ）属の細菌を用い
ることができる。[0009] As the heterotrophic bacterium, a heterotrophic bacterium capable of growing on a normal agar medium, a tripticate soy broth (TSB) medium, or a diluted medium thereof can be used. For example, Paracoccus (Paracoccus) can be used.
ccus) genus, Pseudomonas genus, Flavobacterium genus, Acinetobacter genus, Bulkholderia (Burkholder)
ia) Bacteria belonging to the genus Escherichia can be used.
【００１０】また、本発明において、従属栄養細菌又は
その培養液は、１０〜１０4 cells/ｍｌの従属栄養細菌
初期濃度になるように添加するのが好ましい。従属栄養
細菌の初期濃度が１０cells/ｍｌ未満では、従属栄養細
菌を共存させた効果が見られず、１０4 cells/ｍｌを超
えると酸素摂取の競合に関して従属栄養細菌が優位とな
り、アンモニア酸化活性は向上しない。In the present invention, the heterotrophic bacterium or its culture solution is preferably added so as to have an initial concentration of the heterotrophic bacterium of 10 to 10 4 cells / ml. When the initial concentration of the heterotrophic bacterium is less than 10 cells / ml, the effect of coexisting with the heterotrophic bacterium is not seen. When the initial concentration exceeds 10 4 cells / ml, the heterotrophic bacterium becomes dominant with respect to the competition of oxygen uptake, and the ammonia oxidation activity is reduced. Does not improve.
【００１１】本発明によれば、アンモニア酸化細菌の培
養液中に従属栄養細菌の培養ろ液を添加してもアンモニ
ア酸化細菌の増殖促進効果が得られる。このメカニズム
については、未だ解明されていないが、従属栄養細菌の
培養ろ液を添加する場合、添加後のアンモニア酸化細菌
の培養培地に占める培養ろ液の割合が０．５〜５０容量
％となるように添加するのが好ましい。培養ろ液の割合
が０．５容量％未満であると、アンモニア酸化細菌の増
殖促進効果が認められず、５０容量％を超えると、ろ液
中に残存する有機物による阻害的な効果が現れるおそれ
がある。According to the present invention, the effect of promoting the growth of ammonia-oxidizing bacteria can be obtained even when the culture filtrate of heterotrophic bacteria is added to the culture solution of ammonia-oxidizing bacteria. Although this mechanism has not been elucidated yet, when a culture filtrate of heterotrophic bacteria is added, the ratio of the culture filtrate in the culture medium of the ammonia-oxidizing bacteria after the addition is 0.5 to 50% by volume. It is preferable to add them. If the ratio of the culture filtrate is less than 0.5% by volume, the effect of promoting the growth of ammonia-oxidizing bacteria is not recognized, and if it exceeds 50% by volume, an inhibitory effect due to the organic matter remaining in the filtrate may appear. There is.
【００１２】[0012]
【実施例】次に、本発明を実施例に基づいて詳細に説明
するが、本発明はこれらによって制限されるものではな
い。Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
【００１３】実施例１ アンモニア濃度２８ｍｇ−Ｎ／Ｌのアンモニア酸化細菌
の継代培養用無機培地に、従属栄養細菌〔パラコッカス
・デニトリフィカンス（Paracoccus denitrificans）及
びシュウドモナス〕を１／１００濃度のＴＳＢ培地で３
日間培養した培養液（菌体懸濁液）を２容量％添加した
後、低濃度アンモニア適応種のアンモニア酸化細菌の培
養液（菌体懸濁液）を接種し、アンモニア酸化細菌の培
養を行った。このときの亜硝酸濃度の経日変化を図１に
示す。図１から分かるとおり、従属栄養細菌無添加（ア
ンモニア酸化細菌のみ）の場合にはアンモニア酸化細菌
増殖の誘導期間が４０日以上であったが、従属栄養細菌
の共存下では３１〜３７日に短縮することができ、さら
に誘導期間終了後は亜硝酸生成が著しく少なく、アンモ
ニア酸化細菌の増殖がほとんど行われなかったと推定さ
れるが、従属栄養細菌の共存下では亜硝酸生成速度が急
激に増加し、アンモニア酸化細菌の増殖が急激に向上し
た。Example 1 Heterotrophic bacteria (Paracoccus denitrificans and Pseudomonas) were added to an inorganic medium for subculturing ammonia-oxidizing bacteria having an ammonia concentration of 28 mg-N / L at a concentration of 1/100 TSB. 3 in medium
After adding 2% by volume of a culture solution (cell suspension) cultured for one day, a culture solution (cell suspension) of a low-concentration ammonia-adaptive species of ammonia-oxidizing bacteria is inoculated, and the ammonium-oxidizing bacteria are cultured. Was. FIG. 1 shows the daily change of the nitrite concentration at this time. As can be seen from FIG. 1, the induction period of the growth of ammonia-oxidizing bacteria was 40 days or more in the case where no heterotrophic bacteria were added (only ammonia-oxidizing bacteria), but was shortened to 31 to 37 days in the presence of heterotrophic bacteria. After the induction period, nitrite production was remarkably low, and it was presumed that the growth of ammonia-oxidizing bacteria hardly occurred.However, in the presence of heterotrophic bacteria, the nitrite production rate increased rapidly. However, the growth of ammonia-oxidizing bacteria improved rapidly.
【００１４】実施例２ 従属栄養細菌（パラコッカス・デニトリフィカンス及び
シュウドモナス）を１／１００濃度のＴＳＢ培地で３日
間培養した培養液を孔径０．２２μｍのメンブランフィ
ルターで無菌的にろ過し、このろ液を低濃度アンモニア
適応種のアンモニア酸化細菌の継代培養用培地に予め１
０容量％添加し、低濃度アンモニア適応種のアンモニア
酸化細菌の培養液を接種し、アンモニア酸化細菌の培養
を行った。このときの亜硝酸濃度の経日変化を図２に示
す。図２から分かるとおり、従属栄養細菌無添加（アン
モニア酸化細菌のみ）の場合にはアンモニア酸化細菌増
殖の誘導期間が２０日前後であったが、従属栄養細菌の
共存下では３日〜１週間程度に短縮された。Example 2 A culture obtained by culturing heterotrophic bacteria (Paracoccus denitrificans and Pseudomonas) in a 1/100 concentration TSB medium for 3 days was aseptically filtered through a membrane filter having a pore size of 0.22 μm. The filtrate is preliminarily added to a medium for subculture of ammonia-oxidizing bacteria adapted to low-concentration ammonia.
0% by volume was added, and a culture solution of a low-concentration ammonia-adapted species of ammonia-oxidizing bacteria was inoculated to culture the ammonia-oxidizing bacteria. FIG. 2 shows the daily change of the nitrite concentration at this time. As can be seen from FIG. 2, in the case where no heterotrophic bacterium was added (only the ammonia-oxidizing bacterium), the induction period of the growth of the ammonia-oxidizing bacterium was around 20 days, but in the presence of the heterotrophic bacterium, about 3 days to 1 week. Was shortened to
【００１５】実施例３ トリクロロエチレンに汚染された土壌及び地下水を浄化
するため、アンモニア酸化細菌を大量培養し、土壌及び
地下水中に供給する装置において培養実験を行った。ア
ンモニア酸化細菌培養槽に、従属栄養細菌（パラコッカ
ス・デニトリフィカンス）を初期接種濃度が１０3 cell
s/mlとなるように、培養開始時に接種した。アンモニア
酸化細菌培養槽は、ケモスタット式の連続培養を行って
おり、培養槽内で増殖したアンモニア酸化細菌は、流出
培養液とともに系外に排出される。培養槽への供給原水
は、アンモニア濃度２８ｍｇ−Ｎ／Ｌの無機原水である
ため、従属栄養細菌はアンモニア酸化細菌の代謝物、あ
るいは死んだ菌体を消費して生育すると考えられる。生
菌数の経日変化を図３に示す。図３から、アンモニア酸
化細菌と従属栄養細菌は、やがて一定の菌数で平衡状態
に達するが、アンモニア酸化細菌の生菌数が最大になる
までの期間が、アンモニア酸化細菌単独のときより従属
栄養細菌の共存下で短縮されることが分かる。また、ア
ンモニア酸化細菌の最大生菌数は、従属栄養細菌の共存
下で高くなった。Example 3 In order to purify soil and groundwater contaminated with trichlorethylene, a large amount of ammonia-oxidizing bacteria were cultured and a culture experiment was carried out in an apparatus for supplying the soil and groundwater. The initial inoculation concentration of heterotrophic bacteria (Paracoccus denitrificans) in an ammonia oxidizing bacteria culture tank is 10 3 cells.
It was inoculated at the start of the culture so that the concentration was s / ml. The ammonia-oxidizing bacteria culture tank performs a chemostat-type continuous culture, and the ammonia-oxidizing bacteria grown in the culture tank are discharged out of the system together with the outflow culture solution. Since the raw water supplied to the culture tank is inorganic raw water having an ammonia concentration of 28 mg-N / L, it is considered that the heterotrophic bacteria grow by consuming metabolites of the ammonia-oxidizing bacteria or dead cells. FIG. 3 shows the daily change of the viable cell count. From FIG. 3, the ammonia-oxidizing bacteria and heterotrophic bacteria reach an equilibrium state with a certain number of bacteria, but the time until the viable number of ammonia-oxidizing bacteria reaches the maximum is longer than that of the ammonia-oxidizing bacteria alone. It can be seen that it is shortened in the presence of bacteria. In addition, the maximum viable bacterial count of ammonia-oxidizing bacteria increased in the presence of heterotrophic bacteria.
【００１６】実施例４ 低濃度アンモニア適用種のアンモニア酸化細菌を対象に
ＭＰＮ法で生菌数を計測する際に、通常は４〜８週間の
培養期間を必要とし、無機培地中でアンモニア酸化細菌
のみが存在する場合、増殖に極端に長い時間を要した
り、時には全く増殖が見られないこともあるため、一般
的なＭＰＮ法によって計測された生菌数は過小評価され
る可能性がある。これに対し、ＭＰＮに用いる培地中に
予め微量（初期濃度として１０2 cells/ml）の従属栄養
細菌（パラコッカス・デニトリフィカンス）を混入させ
ておき、培養を行った。この結果、生菌数を計測するた
めの培養を、従来の４〜８週間から２〜４週間に短縮す
ることが可能となり、計測値も従来法より大きかった。
培養期間のＭＰＮ法による生菌数を表１に示す。Example 4 When the number of viable cells is measured by the MPN method for ammonia-oxidizing bacteria to which low-concentration ammonia is applied, a culture period of usually 4 to 8 weeks is required. In the case where only bacteria are present, the growth takes an extremely long time or sometimes no growth is observed at all, so that the number of viable cells measured by the general MPN method may be underestimated. . On the other hand, a small amount (10 2 cells / ml as an initial concentration) of heterotrophic bacteria (Paracoccus denitrificans) was previously mixed in the medium used for MPN, and cultivation was performed. As a result, the culture for measuring the number of viable cells could be shortened from 4 to 8 weeks in the past to 2 to 4 weeks, and the measured value was larger than in the conventional method.
Table 1 shows the viable cell count by the MPN method during the culture period.
【００１７】[0017]
【表１】 [Table 1]
【００１８】[0018]
【発明の効果】本発明により、低濃度アンモニア適応種
のアンモニア酸化細菌の回分的又は連続的培養法及び培
養を伴う菌数計測方法において、従属栄養細菌、その培
養液及び培養ろ液をアンモニア酸化細菌の培養液中に添
加することにより、アンモニア酸化細菌の増殖速度を向
上させ、また、安定したアンモニア酸化細菌の増殖を可
能にすることができる。さらに本発明によれば、増殖の
誘導期を短縮することにより、培養期間の短縮と、これ
に伴う菌数計測に必要な培養期間を短縮することができ
る。Industrial Applicability According to the present invention, in a batch or continuous culture method of a low-concentration ammonia-adapted species of ammonia-oxidizing bacteria and a method for counting the number of bacteria accompanying the culture, the heterotrophic bacteria, the culture solution thereof, and the culture filtrate are subjected to ammonia oxidation. By adding it to the culture solution of bacteria, the growth rate of ammonia-oxidizing bacteria can be improved, and stable growth of ammonia-oxidizing bacteria can be achieved. Furthermore, according to the present invention, by shortening the induction period of proliferation, the culture period can be shortened, and the culture period required for the measurement of the number of bacteria can be shortened.
【図１】実施例１における亜硝酸濃度の経日変化を示す
グラフ図である。FIG. 1 is a graph showing the daily change of nitrite concentration in Example 1.
【図２】実施例２における亜硝酸濃度の経日変化を示す
グラフ図である。FIG. 2 is a graph showing the daily change of nitrous acid concentration in Example 2.
【図３】実施例３における生菌数の経日変化を示すグラ
フ図である。FIG. 3 is a graph showing the change over time in the number of viable bacteria in Example 3.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ (Ｃ１２Ｎ 1/20 Ｃ１２Ｒ 1:38） ──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. 6 Identification code FI (C12N 1/20 C12R 1:38)
Claims (7)
受け、低濃度アンモニアに親和性の高い、基質感受性の
アンモニア酸化細菌を培養するため、該アンモニア酸化
細菌の培養液中に従属栄養細菌、その培養液又は培養ろ
液を添加することを特徴とするアンモニア酸化細菌の培
養法。1. A method for culturing a substrate-sensitive ammonia-oxidizing bacterium whose growth is inhibited by high-concentration ammonia and which has a high affinity for low-concentration ammonia. A method for culturing ammonia-oxidizing bacteria, comprising adding a liquid or a culture filtrate.
養細菌又はその培養液を、従属栄養細菌の初期濃度が１
０〜１０4 cells/ｍｌになるように添加する請求項１記
載のアンモニア酸化細菌の培養法。2. The method according to claim 1, wherein the heterotrophic bacterium or the culture solution thereof is contained in a culture solution of the ammonia-oxidizing bacterium.
The method for cultivating ammonia-oxidizing bacteria according to claim 1, wherein the bacterium is added so as to be 0 to 10 4 cells / ml.
養細菌の培養ろ液を０．５〜５０容量％添加する請求項
１記載のアンモニア酸化細菌の培養法。3. The method for culturing ammonia-oxidizing bacteria according to claim 1, wherein 0.5 to 50% by volume of a culture filtrate of heterotrophic bacteria is added to the culture solution of ammonia-oxidizing bacteria.
Tripticate Soy Broth （ＴＳＢ）培地又はこれらを希
釈した培地に生育することが可能な従属栄養細菌を用い
る請求項１記載のアンモニア酸化細菌の培養法。4. As a heterotrophic bacterium, an ordinary agar medium or
The method for culturing ammonia-oxidizing bacteria according to claim 1, wherein a heterotrophic bacterium capable of growing on a tripticate soy broth (TSB) medium or a medium obtained by diluting them is used.
racoccus）属、シュウドモナス（Pseudomonas ）属、フ
ラボバクテリウム（Flavobacterium）属、アシネトバク
ター（Acinetobacter ）属、バルクホルデリア（Burkho
lderia）属、エシェリヒア（Escherichia ）属の細菌を
用いる請求項１記載のアンモニア酸化細菌の培養法。5. Heterotrophic bacteria, Paracoccus (Pa)
racoccus genus, Pseudomonas genus, Flavobacterium genus, Acinetobacter genus, Bulk horderia (Burkho)
2. The method for cultivating ammonia-oxidizing bacteria according to claim 1, wherein a bacterium belonging to the genus Escherichia is used.
受け、低濃度アンモニアに親和性の高い、基質感受性の
アンモニア酸化細菌の菌数計測を最確値法によって行う
ため、該アンモニア酸化細菌の培養液中に従属栄養細
菌、その培養液又は培養ろ液を添加することを特徴とす
るアンモニア酸化細菌の菌数計測方法。6. Since the growth is inhibited by high-concentration ammonia and the number of substrate-sensitive ammonia-oxidizing bacteria having a high affinity for low-concentration ammonia is measured by the most probable value method, it is necessary to use A method for counting the number of ammonia-oxidizing bacteria, comprising adding a heterotrophic bacterium, a culture solution or a culture filtrate thereof.
養細菌又はその培養液を、従属栄養細菌の初期濃度が１
０〜１０4 cells/ｍｌになるように添加する請求項６記
載のアンモニア酸化細菌の菌数計測方法。7. The method according to claim 7, wherein the heterotrophic bacterium or the culture thereof is contained in a culture solution of the ammonia-oxidizing bacterium.
7. The method for counting the number of ammonia-oxidizing bacteria according to claim 6, wherein the method is added so as to be 0 to 10 4 cells / ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8296098A JPH11253153A (en) | 1998-03-13 | 1998-03-13 | Cultivation of ammonia oxidative bacterium and measurement of number of bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8296098A JPH11253153A (en) | 1998-03-13 | 1998-03-13 | Cultivation of ammonia oxidative bacterium and measurement of number of bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH11253153A true JPH11253153A (en) | 1999-09-21 |
Family
ID=13788804
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8296098A Pending JPH11253153A (en) | 1998-03-13 | 1998-03-13 | Cultivation of ammonia oxidative bacterium and measurement of number of bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH11253153A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006246847A (en) * | 2005-03-14 | 2006-09-21 | Hitachi Plant Technologies Ltd | Method and device for culturing anaerobic ammonia-oxidizing bacterium |
| JP2006289346A (en) * | 2005-03-15 | 2006-10-26 | Hitachi Plant Technologies Ltd | Method and equipment for cultivating anaerobic ammonium-oxidizing bacteria |
| KR100644854B1 (en) * | 2005-10-29 | 2006-11-14 | 한국과학기술연구원 | Method and apparatus for enriching anaerobic ammonium oxidizing bacteria continuously |
| US7960171B2 (en) | 2005-03-14 | 2011-06-14 | Hitachi Plant Technologies, Ltd. | Method and equipment for cultivating anaerobic ammonium-oxidizing bacteria |
-
1998
- 1998-03-13 JP JP8296098A patent/JPH11253153A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006246847A (en) * | 2005-03-14 | 2006-09-21 | Hitachi Plant Technologies Ltd | Method and device for culturing anaerobic ammonia-oxidizing bacterium |
| JP4626884B2 (en) * | 2005-03-14 | 2011-02-09 | 株式会社日立プラントテクノロジー | Culture method and apparatus for anaerobic ammonia oxidizing bacteria |
| US7960171B2 (en) | 2005-03-14 | 2011-06-14 | Hitachi Plant Technologies, Ltd. | Method and equipment for cultivating anaerobic ammonium-oxidizing bacteria |
| JP2006289346A (en) * | 2005-03-15 | 2006-10-26 | Hitachi Plant Technologies Ltd | Method and equipment for cultivating anaerobic ammonium-oxidizing bacteria |
| KR100644854B1 (en) * | 2005-10-29 | 2006-11-14 | 한국과학기술연구원 | Method and apparatus for enriching anaerobic ammonium oxidizing bacteria continuously |
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