JP6182047B2 - Algae growth promoter and algae growth method - Google Patents
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Description
本発明は、藻類の成長促進剤および藻類の増殖方法に関する。 The present invention relates to an algae growth promoter and an algae growth method.
腐植物質は、環境中の物質循環、生物活性、重金属またはＰＰＣＰｓ（日用品由来医薬）の動態に関与しており、結合作用、分散作用、湿潤作用または潤滑作用等様々な機能を有する天然資源である。腐植物質とは、生物の死後、生体有機物が微生物学的または熱化学的作用を受けて崩壊して生じた、化学構造が特定されない有機物（非生体有機物）の総称であり、地球上の水圏、土壌または堆積物環境に広く分布している。腐植物質は、分離法に基づいた操作主義的にフミン酸画分またはフルボ酸画分等に定義され、その構造、分子量または官能基含有量等は、生成される環境によって大きく異なる。 Humic substances are involved in the circulation of substances in the environment, biological activity, heavy metals or PPCPs (drugs derived from daily necessities), and are natural resources that have various functions such as binding, dispersing, wetting or lubrication. . Humic substances are a general term for organic substances (non-biological organic substances) whose chemical structure is not specified, which are generated by the destruction of biological organic substances after the death of a living organism through microbiological or thermochemical effects. Widely distributed in soil or sediment environment. Humic substances are defined in the humic acid fraction or fulvic acid fraction based on the separation method in terms of operation, and the structure, molecular weight, functional group content, and the like vary greatly depending on the environment in which they are produced.
水溶性ガス田施設では、地下５００ｍ以深から地下水（かん水）が汲み上げられ、そこに溶解しているメタンまたはヨードは分離・回収され、販売されている。これらが回収されたかん水は地盤沈下防止のために一部は地下に還元圧入されている。また、かん水は還元圧入のために施設内のサクションタンクに一時貯蔵されているが、そこには藻類の増殖に必要な成分が少なく、増殖し難い環境条件にある。 In the water-soluble gas field facility, groundwater (brine) is pumped from a depth of 500m or below, and dissolved methane or iodine is separated, recovered and sold. Some of the collected brine is reduced and injected underground to prevent land subsidence. In addition, brine is temporarily stored in the suction tank in the facility for reduction injection, but there are few components necessary for the growth of algae, and it is in an environmental condition that is difficult to grow.
しかし、サクションタンク内には藻類ブルームの形成が確認されている。かん水には、藻類ではないが、イネなどの大型植物の成長を促進する効果を有する物質（腐植物質）の存在が確認されている。すなわち、かん水には植物の増殖を促進する効果を有する物質が含まれ、これらが藻類の増殖に効果がある可能性が考えられる。 However, the formation of algal blooms in the suction tank has been confirmed. In the brine, the presence of substances (humic substances) that are not algae but have the effect of promoting the growth of large plants such as rice has been confirmed. That is, the brine contains substances that have an effect of promoting the growth of plants, and these may be effective for the growth of algae.
また、藻類を増殖させる別の方法も知られている。例えば、特許文献１には、藻細胞分裂の速度および藻細胞数を増加させる、藻類の生成物生成に最適化された、フルボ酸およびフミン酸を使用する培養方法が記載されている。
Another method for growing algae is also known. For example,
特許文献２には、フルボ酸およびフミン酸等の植物成長調節剤（成長ホルモン、インドール酢酸等）およびホルモン模倣体（フェノキシ酢酸化合物等）の使用により、独立栄養的、従属栄養的、または光従属栄養的に生育した藻類からのバイオマスの生成を向上させる方法が記載されている。
特許文献３には、かん水に含まれる腐植物質から製造されたフルボ酸は、従来のフルボ酸以上に、植物の生長を促進するのみならず、発芽を促進し、耐病性を向上し、耐乾燥性を向上し、光合成を促進し、代謝を促進し、植物性酵素を活性化することが開示されている。
In
しかしながら、腐植物質の成分であるフルボ酸のどのような成分が、最も効果的に藻類を増殖させるかについては不明な点が多い。一方、藻類によるバイオ燃料生産においては、大量の藻類を時間的およびエネルギー効率的に増殖させることが事業性を左右するため、増殖速度の速い藻類の探索または育種、藻類の成長促進剤およびフォトバイオリアクターの改変等様々な検討が行われている（特許文献４、非特許文献１）。
However, there are many unclear points as to what kind of fulvic acid, which is a component of humic substances, will most effectively grow algae. On the other hand, in biofuel production by algae, timely and energy efficient propagation of a large amount of algae influences the business, so search or breeding of algae with a fast growth rate, algae growth promoter and photobio Various studies such as modification of the reactor have been performed (
したがって、本発明は、藻類の増殖を効果的に促進する成長促進剤を提供することを目的とする。 Therefore, an object of the present invention is to provide a growth promoter that effectively promotes the growth of algae.
本発明者らは、かん水に含有される特定の物性を有するフルボ酸含有組成物が藻類の増殖を効果的に促進することを見出し、本発明を完成させた。 The present inventors have found that a fulvic acid-containing composition having specific physical properties contained in brackish water effectively promotes the growth of algae, and has completed the present invention.
すなわち、本発明は、下記の通りである。
１．かん水から調製したフルボ酸含有組成物を含有する藻類の成長促進剤であって、該フルボ酸含有組成物の分子径が０．４５μｍ以下であり、且つ元素分析値が、Ｃ（３２±１０％）、Ｎ（２．０±１０％）の範囲にあり、Ｃ／Ｎが１５以上である藻類の成長促進剤。
２．前記フルボ酸含有組成物が、分子径が０．０２５μｍ以下であり、且つ分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物を含有する前項１に記載の藻類の成長促進剤。
３．前記フルボ酸含有組成物が以下の（ａ）〜（ｃ）のいずれか１である前項１に記載の藻類の成長促進剤。
（ａ）分子量１０，０００〜３０，０００でＵＶ吸収を持たないフルボ酸含有組成物
（ｂ）分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物
（ｃ）分子量３００以上５００未満でＵＶ吸収が相対的に強いフルボ酸含有組成物
４．前記藻類が、配列番号１で示される１６Ｓ ｒＲＮＡ配列を有する前項１〜３のいずれか１項に記載の藻類の成長促進剤。
５．前記かん水が、地殻変動により地中に封鎖された海水、周辺の地層から溶出した塩分を含有する地下水、淡水に比べて塩分濃度が高い湧水、淡水に比べて塩分濃度が高い温泉水、油層水、および油田、ガス田または炭田の坑廃水から選ばれる少なくとも１である前項１〜４のいずれか１項に記載の藻類の成長促進剤。
６．かん水を超音波洗浄機で超音波処理し、少なくとも二種類のＭＦ膜および／またはＵＦ膜で分画したフルボ酸含有組成物を、藻類を含有する培養液に成長促進剤として添加する藻類の増殖方法。
That is, the present invention is as follows.
1. An algae growth promoter comprising a fulvic acid-containing composition prepared from brine, wherein the fulvic acid-containing composition has a molecular diameter of 0.45 μm or less and an elemental analysis value of C (32 ± 10% ), A range of N (2.0 ± 10%), and an algae growth promoter having a C / N of 15 or more.
2. 2. The algal growth promoter according to
3. 2. The algal growth promoter according to
(A) A fulvic acid-containing composition having a molecular weight of 10,000 to 30,000 and having no UV absorption (b) A fulvic acid-containing composition having a molecular weight of 500 to 3,000 and having a UV absorption (c) A molecular weight of 300 to less than 500 3. A fulvic acid-containing composition having a relatively strong UV absorption The algae growth promoter according to any one of
5. Seawater sealed in the ground due to crustal deformation, groundwater containing salt eluted from the surrounding strata, spring water with higher salinity compared to fresh water, hot spring water, oil reservoir with higher salinity than
6). Proliferation of algae in which the fulvic acid-containing composition obtained by sonicating the brine with an ultrasonic cleaner and fractionated with at least two types of MF membranes and / or UF membranes is added as a growth promoter to a culture solution containing algae. Method.
本発明の藻類の成長促進剤は、フルボ酸含有組成物を含有することにより、藻類、特に藍藻、緑藻および珪藻の増殖を効果的に促進する。また、かん水に本発明の藻類の成長促進剤を添加し、藻類を培養することにより、かん水中の重炭酸イオン、アンモニウムイオン濃度を大幅に減少させることが可能となり、還元井の閉塞の予防策として有効である。 The algae growth promoter of the present invention effectively promotes the growth of algae, particularly cyanobacteria, green algae and diatoms, by containing a fulvic acid-containing composition. In addition, by adding the algae growth promoter of the present invention to brine and culturing the algae, it is possible to greatly reduce the concentration of bicarbonate ions and ammonium ions in the brine, and prevent the blocking of the reduction well. It is effective as
本発明の藻類の成長促進剤に含まれるフルボ酸を含有する組成物（以下、本発明に用いるフルボ酸含有組成物ともいう）は、腐植物質を豊富に含み特定の特性を有するかん水を選択し、該かん水の液性を酸性とし、得られた酸性水から多孔性吸着剤が充填されたカラムにより精製した後、少なくとも二種類のＭＦ膜および／またはＵＦ膜で分画する方法を採用することにより、実用レベルで製造することができる。 A composition containing fulvic acid contained in the algal growth promoter of the present invention (hereinafter also referred to as a fulvic acid-containing composition used in the present invention) is selected from brine containing abundant humic substances and having specific characteristics. , Adopting a method in which the liquidity of the brine is made acidic, and the resulting acidic water is purified by a column packed with a porous adsorbent and then fractionated with at least two types of MF membranes and / or UF membranes. Thus, it can be manufactured at a practical level.
本発明において「フルボ酸」とは、ＩＨＳＳ（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｈｕｍｉｃ Ｓｕｂｓｔａｎｃｅ Ｓｏｃｉｅｔｙ；国際腐植物質学会）法（Ｔｈｕｒｍａｎ ａｎｄ Ｍａｌｃｏｌｍ，１９８１）により、かん水の液性を酸性にして、フミン酸などの酸不溶な腐植物質の画分と、酸可溶な画分とを分離した後、濾過または遠心法などにより酸不溶成分を除去した脱離液を陽イオン交換樹脂等の多孔性吸着剤に吸着させて分離した画分を意味する。 In the present invention, the term “fulvic acid” refers to the acidity of brackish water and the insoluble humic substances such as humic acid by the IHSS (International Humic Society Society) method (Thurman and Malcolm, 1981). And the acid-soluble fraction, and then the adsorbed liquid from which the acid-insoluble components have been removed by filtration or centrifugation is adsorbed on a porous adsorbent such as a cation exchange resin. Means minutes.
かん水とは淡水に比べて塩分濃度の高い地下水を言う。かん水としては、例えば、地殻変動により地中に封鎖された海水、周辺の地層から溶出した塩分を含有する地下水、淡水に比べて塩分濃度が高い湧水、淡水に比べて塩分濃度が高い温泉水、油層水、または油田、ガス田若しくは炭田の坑廃水が挙げられる。しかしながら、原料となるかん水としては、腐植物質を多量に含有しているものが好ましく、腐植物質を多量に含有しているかん水は一般に着色している。 Brine is groundwater with a higher salinity than fresh water. Examples of brackish water include seawater sealed in the ground due to crustal deformation, groundwater containing salt eluted from the surrounding strata, spring water with higher salinity than fresh water, and hot spring water with higher salinity than fresh water. Oil reservoir water, or oilfield, gas field or coalfield mine drainage. However, the brine used as a raw material preferably contains a large amount of humic substance, and the brine containing a large amount of humic substance is generally colored.
なお、かん水の含有物および含有物濃度は産出される地域により大きく異なる。また、同じ地域から産出されるかん水の場合も、採取する深度によって、含有物および含有物濃度は大きく異なる。従って、十分な品質のフルボ酸含有組成物を十分な生産性で得るためには、使用するかん水を注意深く選択する必要がある。 The contents and concentration of brine are greatly different depending on the region where they are produced. In addition, in the case of brines produced from the same area, the inclusions and inclusion concentrations vary greatly depending on the depth of collection. Therefore, in order to obtain a sufficient quality fulvic acid-containing composition with sufficient productivity, it is necessary to carefully select the brine used.
上記の様なかん水の中には、腐植物質の大部分がフルボ酸であるものが存在し、この様なかん水を用いれば、実質的にフルボ酸のみを含有するフルボ酸含有組成物を、安価に製造できるので好ましい。具体的には、腐植物質中のフルボ酸含有量が７０質量％以上であるかん水が好ましく、８０質量％以上がより好ましく、９０質量％以上が更に好ましい。 Among such brines, some of the humic substances are fulvic acid, and if such brines are used, a fulvic acid-containing composition containing substantially only fulvic acid can be obtained at low cost. It is preferable because it can be manufactured. Specifically, brine having a fulvic acid content in the humic substance of 70% by mass or more is preferable, 80% by mass or more is more preferable, and 90% by mass or more is more preferable.
また、上記の様なかん水の中には、０．０９８ＭＰａ（１ａｔｍ）下で２５℃の水に溶解する飽和溶解度以上のメタンと、５０質量ｐｐｍ以上のヨウ素とを含有するものがある。この様なかん水としては、水溶性天然ガス付随かん水などを挙げることができる。 Some brines as described above contain methane having a saturation solubility or higher that dissolves in water at 25 ° C. under 0.098 MPa (1 atm) and iodine having 50 ppm by mass or more. Examples of such brine include water-soluble natural gas-attached brine.
上記の様なかん水からフルボ酸含有組成物を製造する際に、かん水を脱気処理してメタンを初めとする天然ガス成分を除去した後に得られるかん水、または、少なくとも酸化処理しヨウ素を除去して得られる二次かん水を使用することにより、藻類の成長促進剤として高活性なフルボ酸含有組成物が得られると考えられる。 When producing a fulvic acid-containing composition from the brine as described above, the brine is obtained by degassing the brine to remove natural gas components such as methane, or at least oxidizing to remove iodine. It is considered that a highly active fulvic acid-containing composition as an algae growth promoter can be obtained by using the secondary brine obtained in this way.
二次かん水は、酸化処理中にフルボ酸の一部が分解され低分子量化され、藻類に対する吸収効率が向上することができると考えられる。また、フルボ酸の分解により生成した低分子化合物が更に反応および重合などして生じた化合物が関与しているとも考えられる。更に、酸化処理中に活性な官能基がフルボ酸およびフルボ酸分解物に導入される等の化学反応も考えられる。加えて、かん水に含まれるヨウ素および酸化処理で使用される塩素などに起因している可能性もある。 Secondary brine is considered to be able to improve the absorption efficiency for algae because a part of fulvic acid is decomposed during the oxidation treatment to lower the molecular weight. In addition, it is considered that a low molecular weight compound produced by the decomposition of fulvic acid is involved in a compound produced by further reaction and polymerization. Furthermore, a chemical reaction such as an active functional group being introduced into the fulvic acid and the fulvic acid decomposition product during the oxidation treatment is also conceivable. In addition, it may be due to iodine contained in brine and chlorine used in the oxidation treatment.
また、フルボ酸含有組成物を製造する際に、かん水を脱気処理してメタンを初めとする天然ガス成分を除去し、フルボ酸を回収した後にヨウ素を回収することにより、ヨウ素の回収率を向上することができる。 Also, when producing a fulvic acid-containing composition, the brine is degassed to remove natural gas components such as methane, and after recovering fulvic acid, iodine is recovered, thereby improving the iodine recovery rate. Can be improved.
以上で説明した様なフルボ酸含有組成物は、以下に述べる様な特定の方法を採用することにより、実用レベルで製造することができる。 The fulvic acid-containing composition as described above can be produced at a practical level by adopting a specific method as described below.
先ず、かん水を濃縮することが好ましい。濃縮倍率は通常５倍が好ましく、１０倍以上がより好ましく、一方、通常１００倍以下が好ましく、５０倍以下がより好ましい。濃縮方法としては、例えば、減圧蒸発法、吸着法、膜分離法、凍結濃縮法および溶媒抽出法などを挙げることができる。 First, it is preferable to concentrate the brine. The concentration factor is usually preferably 5 times, more preferably 10 times or more, and on the other hand, it is usually preferably 100 times or less and more preferably 50 times or less. Examples of the concentration method include a vacuum evaporation method, an adsorption method, a membrane separation method, a freeze concentration method, and a solvent extraction method.
膜分離法の中でも、精密濾過法、限外濾過法、逆浸透濾過法または透析濾過法などが、濃縮効率が高く、フルボ酸の劣化が少ないと予想され好ましい。 Among membrane separation methods, a microfiltration method, an ultrafiltration method, a reverse osmosis filtration method or a diafiltration method is preferred because it is expected to have high concentration efficiency and little deterioration of fulvic acid.
引続き、かん水の液性を酸性とする。これにより、フミン酸などの酸不溶な腐植物質の画分と、酸可溶なフルボ酸とを分離することができる。具体的には、かん水に塩酸、酢酸またはトリフルオロ酢酸（ＴＦＡ）などを添加してｐＨを所定の値に調整するが、添加する酸が揮発性の酸であることにより脱塩が容易であるので好ましく、フルボ酸の劣化が少ないなどの理由から、塩酸が好ましい。また、ｐＨとしては、十分な分画を行いフルボ酸の劣化を抑制する観点から、１以上が好ましく、一方、５以下が好ましく、４以下がより好ましく、３以下が更に好ましい。 Subsequently, the liquidity of the brine is made acidic. Thereby, an acid-insoluble humic substance fraction such as humic acid and an acid-soluble fulvic acid can be separated. Specifically, hydrochloric acid, acetic acid or trifluoroacetic acid (TFA) is added to brine to adjust the pH to a predetermined value, but desalting is easy because the acid added is a volatile acid. Hydrochloric acid is preferred because it is preferred and there is little deterioration of fulvic acid. Further, the pH is preferably 1 or more from the viewpoint of sufficient fractionation to suppress the deterioration of fulvic acid, while it is preferably 5 or less, more preferably 4 or less, and even more preferably 3 or less.
かん水を酸性とした後、濾過または遠心法などにより酸不溶成分を除去し、酸性水中のフルボ酸を非イオン性多孔質吸着剤に吸着する。 After making the brine water acidic, acid-insoluble components are removed by filtration or centrifugation, and fulvic acid in the acidic water is adsorbed to the nonionic porous adsorbent.
その後、０．１モル／Ｌの水酸化ナトリウムによりフルボ酸を溶出し、再度塩酸等を用いて前記と同様にｐＨを調整する。その後、Ｈ＋型陽イオン交換樹脂によりフルボ酸をナトリウムフリーとし、これを例えば凍結乾燥してフルボ酸を得る。 Thereafter, fulvic acid is eluted with 0.1 mol / L sodium hydroxide, and the pH is adjusted again using hydrochloric acid or the like in the same manner as described above. Thereafter, the fulvic acid is made sodium-free with an H + type cation exchange resin, and this is freeze-dried to obtain fulvic acid.
ここで、原料のかん水がメタン等の天然ガスおよびヨウ素を含んでいる場合、先ず、かん水を脱気して天然ガスを除去する。その後、かん水を酸化処理し、ヨウ素を除去して二次かん水とする。酸化処理によりかん水中のヨウ化物イオンはヨウ素分子に酸化され除去される。なお、酸化処理をすることにより、同時に殺菌処理も行われていると考えられ、滅菌された最終製品を得ることができる。酸化処理の方法としては、例えば、塩素水を添加する方法が挙げられる。 Here, when the raw brine contains natural gas such as methane and iodine, first, the brine is degassed to remove the natural gas. Thereafter, the brine is oxidized and iodine is removed to obtain secondary brine. By the oxidation treatment, iodide ions in the brine are oxidized to iodine molecules and removed. In addition, it is thought that the sterilization process is simultaneously performed by oxidizing, and the sterilized final product can be obtained. Examples of the oxidation treatment method include a method of adding chlorine water.
以上の様にして得られた二次かん水は、かん水の原水の場合と同様に濃縮し、二次かん水の液性を酸性として、二次酸性水からＸＡＤ−８樹脂などの非イオン性多孔質吸着剤により精製することが好ましい。２次かん水からフルボ酸含有組成物を製造することにより、植物活力剤として更に高い活性を有するフルボ酸含有組成物を得ることができる。 The secondary brine obtained as described above is concentrated in the same manner as in the raw water of the brine to make the secondary brine acidic, and from the secondary acidic water to a nonionic porous material such as XAD-8 resin. It is preferable to purify with an adsorbent. By producing a fulvic acid-containing composition from secondary brine, a fulvic acid-containing composition having higher activity as a plant vitality agent can be obtained.
本発明に用いるフルボ酸含有組成物は、かん水を少なくとも二種類のＭＦ膜および／またはＵＦ膜で分画することにより得られる。本発明において使用する濾過膜は、ＭＦ膜（精密濾過膜）は、０．０２５〜０．４５μｍの孔径を有することが好ましい。ＵＦ膜（限外濾過膜）は、０．００１２〜０．００９０μｍの孔径を有することが好ましい。 The fulvic acid-containing composition used in the present invention is obtained by fractionating brine with at least two types of MF membranes and / or UF membranes. As for the filtration membrane used in the present invention, the MF membrane (microfiltration membrane) preferably has a pore diameter of 0.025 to 0.45 μm. The UF membrane (ultrafiltration membrane) preferably has a pore size of 0.0012 to 0.0090 μm.
複数膜を用いた分画は、具体的には、例えば、下記の手法により行う。
１）１回目の濾過は、より大きい孔径の膜を使用してかん水を濾過して濾液を得る。
２）２回目は、一度濾過した濾液を１段階細かいフィルターで再濾過する。
３）再濾過後のフィルターには、１回目に使用した膜と２回目に使用した膜の各孔径間孔径を有する物質が残留する。
４−１）精密濾過（ＭＦ）膜を使用して分画する場合は、孔径０．０２５μｍの濾過膜で濾過したかん水を用いて２回目に濾過したフィルターを逆洗し、残留物質を回収して、１回目に使用した膜と２回目に使用した膜で分画された物質とする。
４−２）限外濾過（ＵＦ）膜を使用する場合、膜の構造上逆洗が出来ないので、新たなかん水に再濾過したフィルターを浸漬し、残留物質を回収し、１回目に使用した膜と２回目に使用した膜で分画された物質とする。
Specifically, fractionation using a plurality of membranes is performed, for example, by the following method.
1) In the first filtration, brine is filtered using a membrane having a larger pore size to obtain a filtrate.
2) At the second time, the filtrate once filtered is refiltered with a one-step fine filter.
3) In the filter after refiltration, substances having pore diameters between the pores of the membrane used for the first time and the membrane used for the second time remain.
4-1) When fractionating using a microfiltration (MF) membrane, backwash the filter that was filtered a second time with brine filtered through a filtration membrane with a pore size of 0.025 μm to recover the residual material. Thus, a substance fractionated by the membrane used the first time and the membrane used the second time is used.
4-2) When an ultrafiltration (UF) membrane is used, backwashing is not possible due to the membrane structure, so the refiltered filter is immersed in fresh brine and the residual material is recovered and used for the first time. The substance is fractionated by the membrane and the membrane used the second time.
なお、かん水は保管中に、増殖促進物質同士が結合して大きな構造を形成する可能性が考えられ、かん水を超音波処理することにより、藻類の増殖を促進する効果が向上し得る。 It should be noted that during the storage of the brine, there is a possibility that the growth promoting substances bind to each other to form a large structure, and the effect of promoting the growth of algae can be improved by ultrasonically treating the brine.
ＭＦ膜およびＵＦ膜の材料としては、例えば、ポリフッ化ビニリデン（ＰＶＤＦ）、酢酸セルロース（ＣＡ）、ポリエチレン（ＰＥ）、ポリスルホン（ＰＳ）、ポリエーテルスルホン（ＰＥＳ）およびセラミックス等が挙げられる。 Examples of materials for the MF film and the UF film include polyvinylidene fluoride (PVDF), cellulose acetate (CA), polyethylene (PE), polysulfone (PS), polyethersulfone (PES), and ceramics.
本発明に用いるフルボ酸含有組成物の元素分析値は、Ｃ（３２±１０％）およびＮ（２．０±１０％）であり、好ましくはＣ（３２±７％）およびＮ（２．０±７％）、より好ましくはＣ（３２±５％）およびＮ（２．０±５％）である。また、Ｃ／Ｎ比は、１５以上であり、好ましくは１７以上である。フルボ酸含有組成物の元素分析は、元素分析装置または理化学分析により行うことができる。 The elemental analysis values of the fulvic acid-containing composition used in the present invention are C (32 ± 10%) and N (2.0 ± 10%), preferably C (32 ± 7%) and N (2.0 ± 7%), more preferably C (32 ± 5%) and N (2.0 ± 5%). The C / N ratio is 15 or more, preferably 17 or more. Elemental analysis of a fulvic acid-containing composition can be performed by an elemental analyzer or physicochemical analysis.
本発明に用いるフルボ酸含有組成物は、分子径が０．４５μｍ以下である。フルボ酸含有組成物の分子径としては、０．００１〜０．０２５μｍ、０．０１２μｍ以下、０．００１μｍ未満の画分が挙げられる。本発明に用いるフルボ酸含有組成物としては、分子径が０．０２５μｍ以下であり、且つ分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物が挙げられる。 The fulvic acid-containing composition used in the present invention has a molecular diameter of 0.45 μm or less. Examples of the molecular diameter of the fulvic acid-containing composition include a fraction of 0.001 to 0.025 μm, 0.012 μm or less, and less than 0.001 μm. Examples of the fulvic acid-containing composition used in the present invention include fulvic acid-containing compositions having a molecular diameter of 0.025 μm or less and having a molecular weight of 500 to 3,000 and UV absorption.
また、本発明に用いるフルボ酸含有組成物としては、以下の（ａ）〜（ｃ）のいずれか１が挙げられる。なお、フルボ酸含有組成物の平均分子量は、ゲルパーメーションクロマトグラフィー法またはサイズ排除クロマトグラフィー法などにより測定できる。
（ａ）分子量１０，０００〜３０，０００でＵＶ吸収を持たないフルボ酸含有組成物
（ｂ）分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物
（ｃ）分子量３００以上５００未満（３００付近）でＵＶ吸収が相対的に強いフルボ酸含有組成物
Moreover, as a fulvic acid containing composition used for this invention, any one of the following (a)-(c) is mentioned. The average molecular weight of the fulvic acid-containing composition can be measured by gel permeation chromatography or size exclusion chromatography.
(A) Fulvic acid-containing composition having a molecular weight of 10,000 to 30,000 and having no UV absorption (b) A fulvic acid-containing composition having a molecular weight of 500 to 3,000 and having a UV absorption (c) Molecular weight of 300 to less than 500 ( Fulvic acid-containing composition with relatively strong UV absorption at around 300)
全分画成分中、前記（ｂ）に属する分画成分は４０〜５０質量％であることが好ましく、数平均分子量は３００以上が好ましく、５００以上がより好ましく、また、５００００以下が好ましく、４００００以下がより好ましい。前記（ｃ）に属する分画成分は３２〜４２質量％であることが好ましい。また、親水性は８０％以上であることが好ましい。 Among the fraction components, the fraction component belonging to (b) is preferably 40 to 50% by mass, the number average molecular weight is preferably 300 or more, more preferably 500 or more, and more preferably 50000 or less, 40000 The following is more preferable. The fraction component belonging to (c) is preferably 32 to 42% by mass. Further, the hydrophilicity is preferably 80% or more.
フルボ酸含有組成物のＵＶ吸収については、ＧＰＣ−ＵＶ／ＴＣ計による分子量分画分析（ＧＰＣ：ゲル・パーメイション・クロマトグラフィー）で分析することができる（特許第２８２８３６７号公報）。 The UV absorption of the fulvic acid-containing composition can be analyzed by molecular weight fraction analysis (GPC: gel permeation chromatography) using a GPC-UV / TC meter (Japanese Patent No. 2828367).
本発明に用いるフルボ酸含有組成物は、２５４ｎｍの紫外吸光／有機炭素が、３ＳＡＣ／ＯＣ（ＨＳ）ｉｎＬ（ｍｇ・ｍ）以下であることが好ましく、２．５ＳＡＣ／ＯＣ（ＨＳ）ｉｎＬ（ｍｇ・ｍ）以下であることがより好ましく、２ＳＡＣ／ＯＣ（ＨＳ）ｉｎＬ（ｍｇ・ｍ）以下であることがさらに好ましい。 The fulvic acid-containing composition used in the present invention preferably has an ultraviolet absorption at 254 nm / organic carbon of 3 SAC / OC (HS) inL (mg · m) or less, and 2.5 SAC / OC (HS) inL (mg M) or less, more preferably 2SAC / OC (HS) inL (mg · m) or less.
ここで、ＳＡＣは紫外吸収検出器（ＵＶＤ、Ｕｌｔｒａ ｖｉｏｌｅｔ ｄｅｔｅｃｔｏｒ）で得られた２５４ｎｍの分光吸収係数［Ｓｐｅｃｔｒａｌ Ａｂｓｏｒｐｔｉｏｎ Ｃｏｅｆｆｉｃｉｅｎｔ（２５４ｎｍ ａｔ １ｍ ｐａｔｈ）］、ＯＣは有機炭素（Ｏｒｇａｎｉｃ ｃａｒｂｏｎ）、ＨＳは腐植物質（Ｈｕｍｉｃｓ）を意味する。 Here, SAC is a spectral absorption coefficient (Spectral Absorption Coefficient (254 nm at 1 m path)) obtained with an ultraviolet absorption detector (UVD, Ultra violet detector), OC is organic carbon (Organic carbon plant), and S is H (Humics).
ＵＶＤにおける応答が芳香族および不飽和構造を反映していることから、ＳＡＣ／ＯＣはＨＳピークの特定の紫外吸光、およびＨＳの芳香族性の尺度となる［Ｓｔｅｆａｎ Ａ．Ｈｕｂｅｒ ｅｔ．ａｌ．，ＷＡＴＥＲ ＲＥＳＥＡＲＣＨ，４５（２０１１），ｐ．８７９−８８５］。 Since the response in UVD reflects aromatic and unsaturated structures, SAC / OC is a measure of the specific UV absorbance of the HS peak, and the aromaticity of HS [Stepan A. et al. Huber et. al. , WATER RESEARCH, 45 (2011), p. 879-885].
フルボ酸含有組成物の形態としては、溶液およびコロイドの少なくとも何れか一方の形態でフルボ酸含有組成物を含有する水性組成物；フルボ酸含有組成物を支持する担持体を含有する分散系組成物；フルボ酸含有組成物を支持する担持体を含有する固形組成物などを挙げることができる。フルボ酸含有水性組成物は、所定量のフルボ酸含有組成物を水および水溶液などに添加することで製造できる。 As a form of the fulvic acid-containing composition, an aqueous composition containing the fulvic acid-containing composition in at least one of a solution and a colloid; a dispersion composition containing a carrier supporting the fulvic acid-containing composition A solid composition containing a carrier that supports the fulvic acid-containing composition. A fulvic acid-containing aqueous composition can be produced by adding a predetermined amount of a fulvic acid-containing composition to water and an aqueous solution.
例えば、少なくとも腐植物質を含み、必要に応じてヨウ素およびメタン等を含むかん水から、フルボ酸含有組成物を単離することなく、フルボ酸の濃度が所定の範囲となるようかん水を濃縮または希釈して、フルボ酸含有水性組成物を製造できる。なお、必要に応じて、過剰の塩類などを除去したり、塩分濃度を低下するために希釈することもある。 For example, the brine is concentrated or diluted so that the concentration of the fulvic acid is within a predetermined range without isolating the fulvic acid-containing composition from the brine containing at least humic substances and optionally iodine and methane. Thus, a fulvic acid-containing aqueous composition can be produced. In addition, as needed, it may dilute in order to remove excess salts or to reduce the salt concentration.
また、少なくとも腐植物質を含み、必要に応じてヨウ素およびメタン等を含むかん水を濃縮する前に、フルボ酸含有組成物の製造の場合と同様に、かん水を脱気して天然ガスを除去し、更に必要に応じて酸化処理してヨウ素を除去し、得られた二次かん水を濃縮または希釈し、過剰の塩類などを除去して、フルボ酸含有水性組成物を製造することもできる。 In addition, before concentrating the brine containing at least humic substances and containing iodine and methane as necessary, the brine is degassed to remove natural gas in the same manner as in the production of the fulvic acid-containing composition, Further, if necessary, it is oxidized to remove iodine, and the obtained secondary brine is concentrated or diluted to remove excess salts and the like to produce a fulvic acid-containing aqueous composition.
本発明に用いるフルボ酸含有組成物はフルボ酸含有水性組成物またはフルボ酸含有分散系組成物とすることにより、例えば、藻類などの植物成長促進剤として使用できる。また、フルボ酸含有組成物がフルボ酸含有固形組成物の場合、該組成物は例えば培地上または耕地中に設置して使用できる。 The fulvic acid-containing composition used in the present invention can be used, for example, as a plant growth promoter for algae, etc. by making it a fulvic acid-containing aqueous composition or a fulvic acid-containing dispersion composition. When the fulvic acid-containing composition is a fulvic acid-containing solid composition, the composition can be used, for example, on a medium or in cultivated land.
本発明において、「藻類」には、原核生物、真核生物を問わず、緑藻類、褐藻類、藍藻類若しくは紅色光合成細菌等の原生動物、または水草等の水生の光合成能を有する生物が含まれる。より具体的には、例えば、光合成緑藻類のクロレラ、バイノス（パラクロレラ属微細藻類パラクロレラ・エスピー・バイノス（Ｐａｒａｃｈｌｏｒｅｌｌａ ｓｐ．ｂｉｎｏｓ）、重油を生産するボトリオコッカスおよびアスタキサンチンを生産することで知られるヘマトコッカスが挙げられる。褐藻類としては、例えば、ワカメまたは昆布等の海草類が挙げられる。藍藻類としては、例えば、スピルリナおよびシネココッカスが挙げられる。 In the present invention, “algae” includes protozoa such as green algae, brown algae, cyanobacteria or red photosynthetic bacteria, or organisms having aquatic photosynthetic ability such as aquatic plants, regardless of whether they are prokaryotes or eukaryotes. . More specifically, for example, the photosynthetic green algae chlorella, binos (Parachlorella sp. Binos), botryococcus producing heavy oil, and hematite known to produce astaxanthin. Examples of brown algae include seaweeds such as seaweed or kelp, etc. Examples of cyanobacteria include spirulina and cinecococcus.
これらの中でも、藍藻類が好ましく、Ｓｙｎｅｃｈｏｃｙｓｔｉｓ属細菌に属する藻類がより好ましく、配列表の配列番号１で示される１６Ｓ ｒＲＮＡ配列を有する藻類が特に好ましい。 Among these, cyanobacteria are preferred, algae belonging to the genus Synechocystis are more preferred, and algae having the 16S rRNA sequence represented by SEQ ID NO: 1 in the sequence listing are particularly preferred.
藍藻、珪藻または緑藻等の脂質を産生する藻類を含有する培養液に、本発明の藻類の成長促進剤を添加することにより、該藻類による脂質の生産量を高めることができる。 By adding the algae growth promoter of the present invention to a culture solution containing algae that produce lipids such as cyanobacteria, diatoms, or green algae, the amount of lipid produced by the algae can be increased.
また、本発明の藻類の成長促進剤を添加してかん水中で藻類を培養することにより、かん水中の重炭酸イオンおよびアンモニウムイオン濃度を大幅に減少させることが可能となり、還元井の閉塞の予防策として有効であり、ガス田の生産効率を向上することができる。 In addition, by adding the algal growth promoter of the present invention and culturing the algae in the brine, it is possible to greatly reduce the concentration of bicarbonate ions and ammonium ions in the brine and prevent the blocking of the reduction well. It is effective as a measure and can improve the production efficiency of the gas field.
なお、本明細書において「有機物質群」または「物質群」と記載することがあるが、それらはフルボ酸を含有する複数の物質の混合物であり、かん水から調製されたフルボ酸を含有する組成物を意味する。 In this specification, it may be referred to as “organic substance group” or “substance group”, which is a mixture of a plurality of substances containing fulvic acid, and a composition containing fulvic acid prepared from brine. Means a thing.
以下に、実施例を挙げて本発明を更に詳細に説明するが、本発明の範囲はそれらに限定されない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited thereto.
＜実施例１＞
（フルボ酸様物質の分析）
日本国千葉県成東地区の地下５００ｍ以深から採取した地下かん水（以下、「かん水Ａ」とする）およびかん水Ａを酸化処理してヨウ素を回収したかん水（以下、「かん水Ｂ」とする）を含む溶液を分析試料とし、フミン酸およびフルボ酸の定量、ＧＰＣ分析、三次元蛍光分析、赤外吸光分析（ＦＴ−ＩＲ）および元素分析を行った。また、４２０ｎｍの吸光度（腐植物質の吸光）を測定した。なお、かん水Ａおよびかん水Ｂによる藻類の成長促進における効果は、実質的に同等であると考えられる。
<Example 1>
(Analysis of fulvic acid-like substances)
Includes underground brine collected from Naruto district of Chiba Prefecture, Japan, below 500m depth (hereinafter referred to as “brine water A”) and brine recovered from iodine by oxidizing the brine water A (hereinafter referred to as “brine water B”). Using the solution as an analysis sample, quantification of humic acid and fulvic acid, GPC analysis, three-dimensional fluorescence analysis, infrared absorption analysis (FT-IR), and elemental analysis were performed. In addition, absorbance at 420 nm (absorption of humic substances) was measured. In addition, it is thought that the effect in the growth promotion of the algae by the brine A and the brine B is substantially equivalent.
試料溶液は濃縮し、アルカリ性として可溶性分を採取し、酸性として不溶分のフミン酸を除去した試料を測定試料とした。比較用試料は「段戸」フミン酸およびフルボ酸を用いた。「段戸」フミン酸およびフルボ酸は愛知県北設楽郡設楽町の段戸褐色森林土壌由来であり、腐植物質学会標準品となっている。 The sample solution was concentrated, a soluble component was collected as alkaline, and a sample from which insoluble humic acid was removed as acidic was used as a measurement sample. As comparative samples, “danto” humic acid and fulvic acid were used. “Stepo” Humic acid and fulvic acid are derived from Dando brown forest soil in Shitara-cho, Kitashirakura-gun, Aichi Prefecture, and are standard products of the Japan Society for Humic Substances.
（分画液のＤＯＣ濃度）
分画液の腐植物質濃度としてＤＯＣ（溶存有機炭素）濃度を測定した結果、ほぼ全ての有機物がフルボ酸分画に画分され、かん水Ａ ＤＯＣ＝４１（ｍｇ／Ｌ）、かん水Ｂ ＤＯＣ＝３５（ｍｇ／Ｌ）となった。
試料 フミン酸分画 フルボ酸分画
かん水Ａ ＜１０ ５４
かん水Ｂ ＜１０ ４４
(DOC concentration of fraction)
As a result of measuring the DOC (dissolved organic carbon) concentration as the humic substance concentration of the fraction solution, almost all organic substances were fractionated into fulvic acid fractions, brine A DOC = 41 (mg / L), brine B DOC = 35. (Mg / L).
Sample Humic acid fraction Fulvic acid fraction Brine A <10 54
Brine B <10 44
吸光光度（段戸標準）での定量値は、分子構造の差異（吸光度の特性）に起因する誤差があると思われるが、下記測定値が得られた。
試料 フミン酸分画 フルボ酸分画
かん水Ａ ＜２ １７
かん水Ｂ ＜２ ２６
Quantitative values in the spectrophotometry (standard standard for terraces) seem to have errors due to differences in molecular structure (absorbance characteristics), but the following measured values were obtained.
Sample Humic acid fraction Fulvic acid fraction Brine A <217
Brine B <226
（ＧＰＣ分析Ｉ）
ＧＰＣ（Ｇｅｌ Ｐｅｒｍｅａｔｉｏｎ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ）−ＵＶ／ＴＣ検出器による分子量分画分析を行い、Ａｓａｈｉｐａｋ ＧＳ−３２０ ２０Ｇ（排除限界：４０，０００Ｄａ分取用、マルチモード）をカラムとして用いてＧＰＣ分析を行った。
(GPC analysis I)
Molecular weight fraction analysis was performed by GPC (Gel Permeation Chromatography) -UV / TC detector, and GPC analysis was performed using Asahipak GS-320 20G (exclusion limit: for 40,000 Da preparative, multimode) as a column.
標準物質による分画特性を表１に示すが、分子量（対数）と保持時間に直線性があるため、出現ピーク位置による推定分子量が計算可能となる。 The fractionation characteristics of the standard substance are shown in Table 1. Since the molecular weight (logarithm) and the retention time are linear, the estimated molecular weight based on the appearance peak position can be calculated.
ＧＰＣ分析の結果、図１（ａ）〜（ｄ）に示すように、かん水Ａおよびかん水Ｂに含まれる有機物質群は、分子量数百〜数万のサイズを持つ物質群（複数物質が混合）が主体である、以下の1〜３に示す物質群（複数の物質の混合物）であった。
１．分子量１０，０００〜３０，０００でＵＶ吸収を持たない有機物質群
２．分子量５００〜３，０００でＵＶ吸収を持つ有機物質群
３．分子量３００付近でＵＶ吸収が相対的に強い有機物質群
As a result of the GPC analysis, as shown in FIGS. 1A to 1D, the organic substance group included in the brine A and the brine B is a substance group having a molecular weight of several hundred to several tens of thousands (mixed with a plurality of substances). Is a substance group (mixture of a plurality of substances) shown in the following 1-3.
1. 1. Organic substance group having a molecular weight of 10,000 to 30,000 and having no
また、かん水Ｂに含まれるフルボ酸は、図２のＦＴ−ＩＲスペクトルに示すように、カルボキシル基などによる親水性が強いことがわかった。 Moreover, it turned out that the fulvic acid contained in the brine B has strong hydrophilicity by a carboxyl group etc. as shown in the FT-IR spectrum of FIG.
（ＧＰＣ分析ＩＩ）
Ｈｕｂｅｒ社ＬＣ−ＯＣＤによる分子量分画分析（ＬＣ−ＯＣＤ：Ｌｉｑｕｉｄ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ−Ｏｒｇａｎｉｃ Ｃａｒｂｏｎ Ｄｅｔｅｃｔｉｏｎ）を行い、ＴＯＳＯ ＴＳＫ ＨＷ−５０Ｓ（排除限界：５０，０００Ｄａ分取用、マルチモード）をカラムとして用いてＧＰＣ分析を行った。
(GPC analysis II)
Molecular weight fractionation analysis (LC-OCD: Liquid Chromatography-Organic Carbon Detection) by Huber LC-OCD was performed, and GPC using TOSO TSK HW-50S (exclusion limit: 50,000 Da preparative, multimode) as a column. Analysis was carried out.
その結果、かん水Ａおよびかん水Ｂに含まれるフルボ酸はカルボキシル基などによる親水性が８０％以上と強く、芳香族含有量は、２．３〜２．６ＳＡＣ／ＯＣ（ＨＳ）ｉｎＬ（ｍｇ・ｍ）であった。 As a result, the fulvic acid contained in brine A and brine B has a strong hydrophilicity of 80% or more due to carboxyl groups, and the aromatic content is 2.3 to 2.6 SAC / OC (HS) inL (mg · m )Met.
（三次元蛍光分析）
フミン酸、フルボ酸の存在を調べるため、かん水Ａおよびかん水Ｂの３次元蛍光分光分析を下記の測定条件で行った。
・測定装置 Ｆｌｕｏｒｏｌｏｇ ３−２２（堀場Ｊｏｂｉｎ Ｙｖｏｎ社製）
・光源 キセノンランプ
・検出器 ＰＭＴ（ｄａｒｋ ｃｏｕｎｔを補正した）
・励起波長 ２５０〜５５０ｎｍ（５ｎｍおき）
・観測波長 ２５０〜６５０ｎｍ（２ｎｍおき）
・スリット幅 励起側２ｎｍ 観測側２ｎｍ
・時定数 ０．２ｓ
・測定モード ＳＣ／ＲＣ（※）各波長の励起光強度で、発光強度を規格化している。
特にフィルターは使用していない。
・溶液測定（光路長１ｃｍ石英セル） ９０°観測
(Three-dimensional fluorescence analysis)
In order to examine the presence of humic acid and fulvic acid, three-dimensional fluorescence spectroscopic analysis of brine A and brine B was performed under the following measurement conditions.
・ Measurement device Fluorolog 3-22 (manufactured by Horiba Jobin Yvon)
・ Light source Xenon lamp ・ Detector PMT (corrected dark count)
・ Excitation wavelength: 250 to 550 nm (every 5 nm)
・ Observation wavelength 250-650nm (every 2nm)
・ Slit width Excitation side 2nm Observation side 2nm
・ Time constant 0.2s
Measurement mode SC / RC (*) The emission intensity is normalized by the excitation light intensity at each wavelength.
In particular, no filter is used.
・ Measurement of solution (quartz cell with optical path length of 1 cm) 90 ° observation
３次元蛍光スペクトルによる測定の結果、かん水Ａおよびかん水Ｂでは、３３０ｎｍ付近の光励起で４００ｎｍ付近に発光している成分が見られ、その濃度はかん水Ａの方が大きいものと考えられた。この発光ピーク位置は、段戸（愛知県北設楽郡設楽町の段戸褐色森林土壌由来、腐植物質学会標準品）（フミン酸）より、段戸（フルボ酸）の方に近かった。 As a result of measurement using a three-dimensional fluorescence spectrum, in brine A and brine B, a component emitting light near 400 nm was observed by light excitation around 330 nm, and the concentration of brine A was considered to be larger. This luminescence peak position was closer to the step door (fulvic acid) than the step door (derived from the step brown forest soil of Shitara Town, Aichi Prefecture Kita Raku-gun, Humic Acid) (humic acid).
しかし、段戸（フルボ酸）では、３２０ｎｍ付近の光励起で４４０ｎｍ付近に発光しており、かん水Ａおよびかん水Ｂに含まれるフルボ酸は段戸（フルボ酸）とは化学構造が異なると思われる。実際、段戸（フルボ酸）の方が、発光強度はかん水Ａおよびかん水Ｂの方が大きかったことからも、その可能性が考えられる。 However, the plate door (fulvic acid) emits light at around 440 nm by photoexcitation at around 320 nm, and the fulvic acid contained in brine A and brine B seems to have a different chemical structure from the terrace door (fulvic acid). In fact, the possibility of the possibility is considered because the luminescence intensity of the step door (fulvic acid) was higher in the brine A and the brine B.
（元素分析）
かん水Ａおよびかん水Ｂに含まれるフルボ酸含有組成物の試料として、かん水Ａおよびかん水ＢをＴｈｅｒｍｏ社製透析膜ＣａｓｓｅｔｔｅＧ２（２，０００ＭＷＣＯ）にて透析し、電気伝導率を、原水の約５（Ｓ／ｍ）から０．０１（Ｓ／ｍ）以下にしたものを、凍結乾燥して元素分析計にて測定した（誤差範囲は、測定値±５％）。その結果を表２に示す。
(Elemental analysis)
As a sample of the fulvic acid-containing composition contained in brine A and brine B, brine A and brine B are dialyzed with Thermo's dialysis membrane Cassette G2 (2,000 MWCO), and the electrical conductivity is about 5 (S / M) to 0.01 (S / m) or less was lyophilized and measured with an elemental analyzer (error range is measured value ± 5%). The results are shown in Table 2.
表２に示すように、かん水Ａおよびかん水Ｂに含まれるフルボ酸含有組成物の元素分析値は、Ｃ（３２±１０％）、Ｎ（２．０±１０％）の範囲にあり、Ｃ／Ｎが１５以上であることがわかった。 As shown in Table 2, the elemental analysis values of the fulvic acid-containing composition contained in brine A and brine B are in the range of C (32 ± 10%) and N (2.0 ± 10%), and C / It was found that N was 15 or more.
＜実施例２＞
（かん水Ｂ中ＤＯＣが藻類の増殖に及ぼす効果）
かん水Ｂ中に含まれる腐植物質が藻類の増殖に及ぼす効果を検討した。かん水Ｂと表３に示すＴＯＣ（全有機炭素）またはＤＯＣを含まない人工かん水（人工培地）とを様々な割合で混合し、かん水Ｂ中の腐植物質（ＤＯＣ濃度）と単離藻類の増殖率との関係を調べた結果を図３に示す。図３に示すように、腐植物質が多い（ＤＯＣ濃度が高い）ほど、単離藻類の増殖率も増加した。
<Example 2>
(Effect of DOC in brine B on algae growth)
The effect of humic substances contained in brine B on the growth of algae was examined. Brine B and TOC (total organic carbon) shown in Table 3 or artificial brine containing no DOC (artificial medium) are mixed in various proportions, and the growth rate of humic substances (DOC concentration) and isolated algae in brine B The result of investigating the relationship with is shown in FIG. As shown in FIG. 3, the more humic substances (the higher the DOC concentration), the greater the growth rate of the isolated algae.
藻類の増殖率は試験開始時と７日間培養後のサンプルの吸光度（７５０ｎｍ）を分光光度計にて測定し、式１を用いて増殖率を算出し評価した。
増殖率（％）＝対象系の吸光度／コントロール系の吸光度×１００％・・・式１
The growth rate of the algae was evaluated by measuring the absorbance (750 nm) of the sample at the start of the test and after culturing for 7 days with a spectrophotometer, and calculating the growth
Growth rate (%) = absorbance of target system / absorbance of control system × 100
なお、コントロール系の吸光度とはかん水Ｂそのものを用いて藻類を７日間培養した後の吸光度であり、対象系の吸光度とは、膜を使った分画操作を加えたかん水Ｂを用いて藻類を７日間培養した後の吸光度である。 The absorbance of the control system is the absorbance after culturing the algae using the brine B itself for 7 days, and the absorbance of the target system is the algae using the brine B to which the fractionation operation using the membrane is added. Absorbance after 7 days of culture.
単離藻類の培養は、サクションタンクから藻類ブルームを回収し、少量をかん水Ｂに植菌し蛍光灯にて光強度５．０±０．５Ｗ／ｍ２にて連続照射した。７日間経過後にはかん水Ｂに適した藻類が優先的に増殖した。この一連の作業を複数回実施することで増殖速度の速い藻叢を集積した。次に、集積した藻叢をＢＧ−１１平板培地上に塗抹し、同培養条件下において生育するコロニーを釣菌した。さらに、コロニーをＰＹＧ培地で培養して共生微生物の有無を確認した。以上の操作により、増殖速度の高い藻類を単離し、当該藻類を供試藻類とした。なお、当該藻類は、形態学的観察より、藍色細菌（藍藻）であると推察された。 For the culture of the isolated algae, the algal bloom was collected from the suction tank, a small amount was inoculated into the brine B and continuously irradiated with a fluorescent lamp at a light intensity of 5.0 ± 0.5 W / m 2 . Algae suitable for brackish water B preferentially grew after 7 days. By carrying out this series of operations a plurality of times, algae flora with a high growth rate was accumulated. Next, the accumulated algal flora was smeared on a BG-11 plate medium, and colonies that grew under the same culture conditions were fished. Furthermore, the colonies were cultured in PYG medium to confirm the presence or absence of symbiotic microorganisms. Through the above operation, an algae with a high growth rate was isolated, and the algae was used as a test algae. The algae were presumed to be cyanobacteria (cyanophyceae) from morphological observation.
前記藻類の１６Ｓ ｒＲＮＡ配列を配列表の配列番号１に示す。前記藻類の菌体生産量、油分含有量、油分成分、油分成分生産性を調べた結果を表４に示す。 The 16S rRNA sequence of the algae is shown in SEQ ID NO: 1 in the sequence listing. Table 4 shows the results of examining the algae cell production, oil content, oil component, and oil component productivity of the algae.
（藻類の同定）
かん水Ｂ中で増殖速度の速い藻類を特定するために、（１）藻類の顕微鏡観察による形態的特徴の確認と、（２）遺伝子解析としてダイレクトシークエンス法による１６Ｓ−ｒＲＮＡ遺伝子配列の解読を実施し、２つのデータを合わせて藻類の特定を行った。
(Identification of algae)
In order to identify algae with a fast growth rate in brine B, (1) confirmation of morphological characteristics by microscopic observation of algae, and (2) decoding of 16S-rRNA gene sequence by direct sequence method as gene analysis The algae was identified by combining the two data.
（１）藻類試料液をスライドグラスに滴下し顕微鏡観察を行い、形態的特徴について確認した結果、緑色のＣｙａｎｏｂａｃｔｅｒｉａ門細菌様の細胞が検出された。細胞形状は単細胞であり、球菌−二連球菌であった。細胞の大きさは、２．６±０．２μｍ（ｎ＝１６）であった。これらの特徴は、Ｃｙａｎｏｂａｃｔｅｒｉａ ｓｕｂｓｅｃｔｉｏｎ Ｉ（Ｃｈｒｏｏｃｏｃｃａｌｅｓ目）の特徴と一致した（Ｒｉｃｈａｒｄ Ｗ．ｅｔ ａｌ．，Ｉｎ，Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ ｓｅｃｏｎｄ ｅｄｉｔｉｏｎ，ｐ．４７３，２００４）。したがって、本試料中に含まれる藻類はＣｙａｎｏｂａｃｔｅｒｉａ ｓｕｂｓｅｃｔｉｏｎ Ｉ（Ｃｈｒｏｏｃｏｃｃａｌｅｓ目）に分類されるものと考えられた。 (1) The algal sample solution was dropped on a slide glass and observed under a microscope, and as a result of confirming the morphological characteristics, green Cyanobacteria bacterium-like cells were detected. The cell shape was single cell and was cocci-deptococci. The cell size was 2.6 ± 0.2 μm (n = 16). These features were consistent with those of Cyanobacterial subsumption I (Chroococcales) (Richard W. et al., In, Bergey's Manual of Systematic second, 3, 4). Therefore, it was considered that the algae contained in this sample are classified as Cyanobacteria subsection I (Chroococcus).
（２）ダイレクトシークエンスによる塩基配列の解析
試料を遠心分離により集菌した後、上清を除去し、滅菌水で数回菌体を洗浄した。洗浄した菌体からＳｏｉｌ ＤＮＡ Ｉｓｏｌａｔｉｎ ｋｉｔ（ＭｏＢｉｏ社製）を用いてＤＮＡを抽出した。抽出方法は説明書に従った。
(2) Analysis of base sequence by direct sequencing After collecting the samples by centrifugation, the supernatant was removed, and the cells were washed several times with sterile water. DNA was extracted from the washed cells using a Soil DNA Isolatin kit (MoBio). The extraction method followed the instructions.
抽出したＤＮＡを鋳型として、藻類（Ｃｙａｎｏｂａｃｔｅｒｉａ門細菌）の１６Ｓ ｒＲＮＡ遺伝子配列に特異的なプライマーを用いてＰＣＲを行った。ＰＣＲの反応条件は、９６℃にて１分間の後に、９６℃にて３０秒間、６０℃にて１分間および７２℃にて１分間を３０サイクル後、７２℃にて１０分間とした。プライマーは既往の報告（Ｎｕｂｅｌ，Ｕ．ｅｔ ａｌ．Ａｐｐｌｉｅｄ ａｎｄ ｅｎｖｉｒｏｎｍｅｎｔａｌ ｍｉｃｒｏｂｉｏｌｏｇｙ ６３：３３２７−３２，１９９７）において、Ｃｙａｎｏｂａｃｔｅｒｉａ門細菌の１６Ｓ ｒＲＮＡ遺伝子配列の特異的な増幅に用いられている表５に示すプライマーを用いた。 Using the extracted DNA as a template, PCR was performed using a primer specific for the 16S rRNA gene sequence of algae (Cyanobacteria). PCR reaction conditions were 96 ° C. for 1 minute, 96 ° C. for 30 seconds, 60 ° C. for 1 minute and 72 ° C. for 1 minute, 30 cycles, and 72 ° C. for 10 minutes. The primers shown in Table 5 used for specific amplification of the 16S rRNA gene sequence of Cyanobacteria phytobacterium in the previous report (Nubel, U. et al. Applied and environmental microbiology 63: 3327-32, 1997) Was used.
試料から抽出したＤＮＡを鋳型として、Ｃｙａｎｏｂａｃｔｅｒｉａ門細菌の１６Ｓ ｒＲＮＡ遺伝子配列を増幅し、塩基配列を決定した。その結果、塩基長９７７ｂｐである配列表の配列番号１に示す配列であった。ＢＬＡＳＴによる相同性解析の結果、解読した塩基配列はＳｙｎｅｃｈｏｃｙｓｔｉｓ ｓｐ．ＰＣＣ ６８０３ ｓｔｒａｉｎ ＧＴ−Ｓと９８％と高い相同性を示した。 Using the DNA extracted from the sample as a template, the 16S rRNA gene sequence of Cyanobacteria bacterium was amplified, and the nucleotide sequence was determined. As a result, it was the sequence shown in SEQ ID NO: 1 in the sequence listing having a base length of 977 bp. As a result of the homology analysis by BLAST, the decoded base sequence is Synechocystis sp. PCC 6803 strain GT-S showed high homology with 98%.
１６Ｓ ｒＲＮＡ遺伝子配列の解析結果から、藻類（Ｃｙａｎｏｂａｃｔｅｒｉａ門細菌）はＳｙｎｅｃｈｏｃｙｓｔｉｓ属細菌に属する藻類である可能性が考えられた。また、顕微鏡観察からはＣｙａｎｏｂａｃｔｅｒｉａ ｓｕｂｓｅｃｔｉｏｎ Ｉに分類される可能性が考えられ、このＣｙａｎｏｂａｃｔｅｒｉａ ｓｕｂｓｅｃｔｉｏｎ Ｉの中には、Ｓｙｎｅｃｈｏｃｙｓｔｉｓ属細菌は含まれている。 From the analysis result of the 16S rRNA gene sequence, it was considered that the algae (Cyanobacteria genus bacteria) may be algae belonging to the genus Synechocystis. In addition, there is a possibility that it is classified into Cyanobacterial subsection I from microscopic observation, and this Cyanobacterial subsection I includes bacteria belonging to the genus Synechocystis.
そこで本試料とＳｙｎｅｃｈｏｃｙｓｔｉｓ属細菌の形態的特徴の比較を行い、種名の特定を行った結果、試料中に含まれる藻類は形態的および遺伝子配列においてもＳｙｎｅｃｏｃｙｓｔｉｓ属細菌に分類された。分子系統樹を作成して種名の特定を試みたところ、本試料に最も近縁な種はＢＬＡＳＴによる相同性解析と同様にＳｙｎｅｃｈｏｃｙｓｔｉｓ ｓｐ．であった。以上の結果から、かん水Ｂ中で増殖速度の速い藻類はＳｙｎｅｃｈｏｃｙｓｔｉｓ ｓｐ．であると考えられた。 Therefore, as a result of comparing the morphological characteristics of this sample and Synechocystis genus bacteria and specifying the species name, the algae contained in the sample were classified as Synecocystis genus bacteria also in morphological and gene sequences. When a molecular phylogenetic tree was created to identify the species name, the species most closely related to this sample was Synchocystis sp., Similar to the homology analysis by BLAST. Met. From the above results, the algae having a fast growth rate in the brine B are Synechocystis sp. It was thought that.
（かん水Ｂの画分が単離藻類の増殖に与える効果）
精密濾過膜であるＭｉｌｌｉｐｏｒｅ社製Ｎｉｔｒｏｃｅｌｌｕｌｏｓｅ Ｍｅｍｂｒａｎｅ（孔径０．４５μｍ、０．２２μｍ、０．１μｍ、０．０５μｍまたは０．０２５μｍ）を用いてかん水Ｂを濾過した。この濾液（画分）を培地として用いることで画分に残留しているかん水Ｂ内の物質が単離藻類の増殖に与えている効果を検討した。また、分子径が０．０２５μｍ以下の物質についても検討を行うため、分子量（Ｍｗ）で分画可能な限外濾過膜であるＭｉｌｌｉｐｏｒｅ社製Ａｍｉｃｏｎ Ｕｌｔｒａ[Ｍｗ：１００，０００（＝孔径０．００９０μｍ）、３０，０００（＝孔径０．００３４μｍ）、３，０００（＝孔径０．００１２μｍ）]を用いてＮｉｔｒｏｃｅｌｌｕｌｏｓｅ Ｍｅｍｂｒａｎｅと同様の方法で培地を調製した。該培地に単離藻類を植菌し増殖に与える効果を確認した。
(Effect of fraction of brine B on growth of isolated algae)
The brine B was filtered using a microfiltration membrane Nitrocellulose Membrane (pore size 0.45 μm, 0.22 μm, 0.1 μm, 0.05 μm or 0.025 μm) manufactured by Millipore. By using this filtrate (fraction) as a medium, the effect of the substances in brine B remaining in the fraction on the growth of isolated algae was examined. In addition, in order to investigate a substance having a molecular diameter of 0.025 μm or less, Amicon Ultra manufactured by Millipore [Mw: 100,000 (= pore diameter 0.0090 μm), which is an ultrafiltration membrane that can be fractionated by molecular weight (Mw). ), 30,000 (= pore diameter 0.0034 μm), 3,000 (= pore diameter 0.0012 μm)], and a medium was prepared in the same manner as in Nitrocellulose Membrane. Inoculated isolated algae in the medium and confirmed the effect on growth.
最初に、かん水Ｂを上記ＭＦ／ＵＦ膜の各々単独膜を用いて濾過し、培養用の濾液を調製し、取り除いた物質の効果を検討した。その結果、図４から分かるように、ネガティブコントロールにおいて、０．００１２μｍ〜０．０２５μｍの大きさを有する物質が単離藻類の増殖を促進する効果を与えていると考えられる。 First, brine B was filtered using each of the above MF / UF membranes to prepare a culture filtrate, and the effects of the removed substances were examined. As a result, as can be seen from FIG. 4, it is considered that a substance having a size of 0.0012 μm to 0.025 μm has an effect of promoting the growth of isolated algae in the negative control.
次に、複数の濾過膜を使用して分画成分を得る方法について説明する。まず、３０ｍｌのかん水Ｂを精密濾過膜であるＭｉｌｌｉｐｏｒｅ社製Ｎｉｔｒｏｃｅｌｌｕｌｏｓｅ Ｍｅｍｂｒａｎｅ（孔径０．４５μｍ、０．２２μｍ、０．１μｍ、０．０５μｍまたは０．０２５μｍ）およびＡｍｉｃｏｎ Ｕｌｔｒａ（孔径０．００９０μｍ、０．００３４μｍまたは０．００１２μｍ）を用いて濾過した。一度濾過した後に１段階細かいフィルターで再濾過した（孔径０．４５μｍで濾過した場合、孔径０．２２μｍで再濾過する）。すると、再濾過後のフィルター（この場合孔径０．２２μｍ）には各孔径間のサイズ（分子径０．２２μｍ〜０．４５μｍ）を有する物質が残留する。 Next, a method for obtaining a fraction component using a plurality of filtration membranes will be described. First, 30 ml of brine B is a microfiltration membrane manufactured by Millipore Nitrocellulose Membrane (pore diameter 0.45 μm, 0.22 μm, 0.1 μm, 0.05 μm or 0.025 μm) and Amicon Ultra (pore diameter 0.0090 μm, 0.005 μm). 0034 μm or 0.0012 μm). After filtering once, it was re-filtered with a one-step fine filter (when filtered with a pore size of 0.45 μm, it was re-filtered with a pore size of 0.22 μm). Then, a substance having a size (molecular diameter of 0.22 μm to 0.45 μm) between the pore diameters remains in the filter after refiltration (in this case, the pore diameter is 0.22 μm).
その後、Ｍｉｌｌｉｐｏｒｅ社製Ｎｉｔｒｏｃｅｌｌｕｌｏｓｅ Ｍｅｍｂｒａｎｅのものは孔径０．０２５μｍで濾過したかん水Ｂを用いて再濾過したフィルターを逆洗し、残留物質を回収した。一方、Ａｍｉｃｏｎ Ｕｌｔｒａのものは逆洗が出来ない構造のため、３０ｍｌの新たなかん水Ｂに再濾過したフィルターを浸漬し、残留物質を回収した。 Thereafter, the Nitrocellulose Membrane manufactured by Millipore backwashed the filter after re-filtering with brine B filtered with a pore size of 0.025 μm, and recovered the residual material. On the other hand, since Amicon Ultra's structure cannot be backwashed, the refiltered filter was immersed in 30 ml of fresh brine B to recover the residual material.
なお、初期かん水Ｂ量を６０ｍｌ、９０ｍｌとして濾液を作成することでＮｉｔｒｏｃｅｌｌｕｌｏｓｅ Ｍｅｍｂｒａｎｅ系は１倍，２倍，３倍濃縮液を、Ａｍｉｃｏｎ Ｕｌｔｒａ系は２倍，３倍，４倍濃縮液を得た。そして、これらの溶液に単離藻類を植菌し、その増殖傾向を観察することで増殖促進物質がどの分子径に存在するか検討を行った。 In addition, by making the filtrate with initial watering B amount of 60 ml and 90 ml, Nitrocellulose Membrane system obtained 1 time, 2 times, 3 times concentrated liquid, Amicon Ultra system obtained 2 times, 3 times, 4 times concentrated liquid. . Then, by inoculating isolated algae in these solutions and observing their growth tendency, the molecular diameter of the growth promoting substance was examined.
膜画分の追加投入による単離藻類の培養実験により、分画物質が単離藻類の増殖に与える効果を検討した結果を図５に示す。 FIG. 5 shows the result of examining the effect of the fractionated substance on the growth of the isolated algae by the culture experiment of the isolated algae by adding the membrane fraction.
膜画分の追加投入による単離藻類の培養実験において、分子径０．００１２μｍ以下の物質（ＵＦ膜で回収）を加えた系では膜画分を加えることで単離藻類の増殖に寄与している可能性が示唆された。また、物質が細かくなるほど増殖への効果が得られた。図５は分子径０．００１２μｍ以下の分画成分が最も増殖率を高めたことを示している。 In a culture experiment of isolated algae by adding the membrane fraction, in a system to which a substance having a molecular diameter of 0.0012 μm or less (collected with a UF membrane) is added, the membrane fraction is added to contribute to the growth of the isolated algae. The possibility was suggested. Moreover, the effect on proliferation was acquired, so that the substance became finer. FIG. 5 shows that the fraction component having a molecular diameter of 0.0012 μm or less has the highest growth rate.
更に、図６はかん水Ｂ中の増殖促進物質を分子径ごとに分画し、その画分を新たなかん水Ｂに追加添加（画分量を増加）したものに、単離藻類を植菌した場合における増殖率を分かりやすく表示している。ただし、孔径０．００１２μｍ、０．００３４μｍまたは０．００９０μｍのフィルターを使用した膜分画はかん水Ｂに追加投入した。 Further, FIG. 6 shows a case where the growth promoting substance in brine B is fractionated by molecular diameter, and the fraction is added to new brine B (increase in the amount of fraction) to inoculate the isolated algae. The growth rate is clearly displayed. However, the membrane fraction using a filter having a pore size of 0.0012 μm, 0.0034 μm or 0.0090 μm was additionally added to brine B.
図６に示すように、分子径０．００９０μｍ以下の物質を加えた系では、画分量を増加（濃縮率が高い）させた全ての系で単離藻類の増殖が促進される傾向が見られた。 As shown in FIG. 6, in the system to which a substance having a molecular diameter of 0.0090 μm or less was added, there was a tendency that the growth of the isolated algae was promoted in all the systems in which the fraction amount was increased (concentration rate was high). It was.
＜実施例３＞
（超音波処理したかん水Ｂ中成分の単離藻類に及ぼす増殖効果）
ガス田より採水したかん水は保管中に、増殖促進物質同士が結合して大きな構造を形成する可能性が考えられる。そこで、かん水Ｂを超音波洗浄機で３８Ｈｚ、６０分間、超音波処理し、各画分で単離藻類を培養、かん水Ｂ中の増殖促進物質の分子径を検討した。すなわち、分散させたかん水Ｂ中の物質が単離藻類の増殖に与える効果を検討した。
<Example 3>
(Proliferation effect on isolated algae of components in brine B subjected to ultrasonic treatment)
It is conceivable that the brine collected from the gas field may form a large structure by combining the growth promoting substances during storage. Therefore, the brine B was sonicated with an ultrasonic cleaner at 38 Hz for 60 minutes, and the isolated algae were cultured in each fraction, and the molecular diameter of the growth promoting substance in the brine B was examined. That is, the effect of dispersed substances in brine B on the growth of isolated algae was examined.
かん水Ｂを超音波破砕し、濾過を行った対象系（超音波破砕のプロット）では分子径０．００１２μｍ、０．００３４μｍおよび０．００９０μｍのいずれの場合でも１００％に近い増殖率が得られた。その結果を図７に示す。図７に示すように、藻類の増殖促進効果を持つ物質は０．００１２μｍ以下の分子径を有する可能性が示唆された。従って、かん水を超音波処理することは、藻類の成長促進剤を調製する方法として優れている。 In the target system (ultrasonication plot) in which the brine B was ultrasonically crushed and filtered, a growth rate close to 100% was obtained in any of the molecular diameters 0.0012 μm, 0.0034 μm, and 0.0090 μm. . The result is shown in FIG. As shown in FIG. 7, it was suggested that a substance having an effect of promoting the growth of algae may have a molecular diameter of 0.0012 μm or less. Therefore, ultrasonic treatment of brackish water is an excellent method for preparing algal growth promoters.
＜実施例４＞
（脂質蓄積能を有する藻類への適用試験）
高脂質蓄積能をもつ３種（藍藻、珪藻、緑藻）の藻類をＮＩＥＳカルチャーコレクション等から数種類ずつ入手、かん水Ｂの膜画分に植菌、培養し、高脂質蓄積能をもつ藻類への適用性を検討し、図８に示す結果を得た。
<Example 4>
(Application test to algae with lipid accumulation ability)
Obtain several kinds of algae with high lipid accumulation ability (Cyanobacteria, Diatom, Green algae) from NIES Culture Collection, etc., inoculate and culture in the membrane fraction of brine B, and apply to algae with high lipid accumulation ability The results shown in FIG. 8 were obtained.
図８に示すように、その結果、超音波処理前と処理後の増殖率で単離藻類と同様の傾向が見られた。従って、かん水Ｂの増殖促進物質は高脂質蓄積能を有する様々な藻類にも適用可能である。すなわち、図８はＸ軸の分子径よりも小さい物質のみを含有する画分に、脂質蓄積能が優れた藻類（藍藻、緑藻、珪藻）を植菌した場合の増殖率を表している。 As shown in FIG. 8, as a result, the same tendency as that of the isolated algae was observed in the growth rate before and after the ultrasonic treatment. Therefore, the growth promoting substance of brine B is applicable to various algae having high lipid accumulation ability. That is, FIG. 8 shows the growth rate when algae (cyanobacteria, green algae, diatoms) excellent in lipid accumulation ability are inoculated into a fraction containing only a substance smaller than the molecular diameter of the X axis.
図８に示すように、分子径０．４５μｍ以下の画分は藻類の増殖促進効果を有することがわかった。また、単離藻類の場合と同様に、超音波破砕を行っていないかん水Ｂの画分では、分子径０．００９０μｍ〜０．０２５μｍの画分に増殖促進物質の存在が示唆されたが、超音波破砕後では分子径０．００１２μｍ以下の物質のみしか含有されていない画分を使用した場合でも目立った増殖率の減少は見られなかった。 As shown in FIG. 8, it was found that the fraction having a molecular diameter of 0.45 μm or less has an algae growth promoting effect. In addition, as in the case of isolated algae, in the fraction of brine B that was not subjected to ultrasonic disruption, the presence of a growth promoting substance was suggested in the fraction with a molecular diameter of 0.0090 μm to 0.025 μm. After sonication, even when a fraction containing only a substance having a molecular diameter of 0.0012 μm or less was used, no significant reduction in the growth rate was observed.
このことから、脂質蓄積能が優れた藻類でも分子径０．００１２μｍ以下の画分にも増殖促進物質があることが示唆された。また、かん水Ｂに含まれる増殖促進物質は藍藻だけでなく、緑藻および珪藻でも同様の効果が得られる可能性が示唆された。 This suggests that there is a growth promoting substance even in algae with excellent lipid accumulation ability even in fractions having a molecular diameter of 0.0012 μm or less. In addition, it was suggested that the growth promoting substance contained in the brine B may be obtained not only by cyanobacteria but also by green algae and diatoms.
更に、カラムクロマト法により測定したかん水Ａの流路壁底面に自生している藻類中の脂質含有量を表６に示す。 Furthermore, Table 6 shows the lipid content in the algae native to the bottom of the channel wall of brine A measured by column chromatography.
表６に示すように、かん水Ａの流路壁底面に自生している藻類マット中の脂質含有量は、日照条件が不利な冬季においても３．８％であった。この結果から、これらの藻類に抽出可能な脂質が存在することが分かり、かん水を活用した粗放的な藻類培養の可能性が示唆された。 As shown in Table 6, the lipid content in the algal mat grown naturally on the bottom of the channel wall of brine A was 3.8% even in the winter when the sunshine conditions were disadvantageous. From these results, it was found that these algae have extractable lipids, suggesting the possibility of extensive algae culture using brine.
＜実施例５＞
（かん水採取時／藻類培養後の培養液の分析結果）
ガス田の管理上、硫酸還元菌等の微生物によるスライム形成（水酸化鉄）が圧入井閉塞の一因となっている。そこで、藻類を培養することで、重炭酸イオン、アンモニウムイオン濃度を大幅に減少させることが可能か否か試験し、還元井の閉塞に対する予防策としての可能性を検討した。
<Example 5>
(At the time of brine collection / analysis result of culture solution after algae culture)
In the management of gas fields, slime formation (iron hydroxide) by microorganisms such as sulfate-reducing bacteria contributes to blockage of the injection well. Therefore, it was tested whether or not the concentration of bicarbonate ions and ammonium ions could be significantly reduced by culturing algae, and the possibility as a preventive measure against reduction well blockage was examined.
試料は、かん水Ａ、かん水Ｂおよび培養後培養液を使用し、ｐＨ、重炭酸イオンおよびアンモニウムイオン濃度を測定した。 The samples used were brine A, brine B and post-culture medium, and the pH, bicarbonate ion and ammonium ion concentrations were measured.
重炭酸イオンおよびアンモニウムイオン濃度の分析結果を表７に示す。表７に示すように、かん水で藻類を培養し、培養後の濾過液を還流させれば還元井の閉塞に対する予防策として有効であることが分かった。 Table 7 shows the analysis results of the bicarbonate ion and ammonium ion concentrations. As shown in Table 7, it was found that culturing algae with brine and refluxing the filtrate after culturing was effective as a preventive measure against blocking of the reduction well.
以上の結果から、かん水中に藻類の増殖促進効果をもたらす物質が含まれていることが確認された。かん水を超音波破砕後、濾過によって得た画分で単離藻類を培養した実験とＧＰＣ分析の結果を比較すると、いずれの結果からも分子径が０．００１２μｍ以下の物質が藻類の増殖に顕著な促進効果を与えていることが示唆された。この分子径の範囲には腐植物質が存在するため、腐植物質が藻類の増殖促進に寄与していることが推察される。 From the above results, it was confirmed that the aqueduct water contains a substance that has an effect of promoting the growth of algae. Comparing the experiment of cultivating isolated algae with the fraction obtained by filtration after ultrasonic pulverization of brine and the results of GPC analysis, from all the results, substances with a molecular diameter of 0.0012 μm or less are prominent in the growth of algae It was suggested that it has a positive effect. Since humic substances exist within this molecular diameter range, it is assumed that the humic substances contribute to the promotion of algae growth.
また、前記物質はガス田由来の単離藻類だけではなく、脂質蓄積能が優れた藻類の増殖にも増殖促進効果を与えていることが確認された。このことから、かん水の利用方法として脂質蓄積に優れた藻類の培養が可能であり、効率的なエネルギー生産が期待される。 In addition, it was confirmed that the substance has a growth promoting effect not only on the isolated algae derived from the gas field but also on the growth of algae with excellent lipid accumulation ability. From this, it is possible to culture algae excellent in lipid accumulation as a method of using brine, and efficient energy production is expected.
本発明の藻類の成長促進剤は、植物の成長促進剤として農業分野での利用が期待される。また、かん水に本発明の藻類の成長促進剤を添加すれば、重炭酸イオン、アンモニウムイオン濃度を大幅に減少させることが可能となり、還元井の閉塞の予防策として有効であり、ガス田の生産効率が向上する。 The algal growth promoter of the present invention is expected to be used in the agricultural field as a plant growth promoter. In addition, if the algal growth promoter of the present invention is added to brine, the concentration of bicarbonate ions and ammonium ions can be greatly reduced, which is effective as a preventive measure for reducing well clogging and production of gas fields. Efficiency is improved.
Claims (3)
該フルボ酸含有組成物は分子径が０．００３４μｍ以下であり、
元素分析値が、Ｃ（３２±１０％）、Ｎ（２．０±１０％）の範囲にあり、Ｃ／Ｎが１５以上であり、
且つ以下の（ｂ）または（ｃ）である藻類の成長促進剤。
（ｂ）分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物
（ｃ）分子量３００以上５００未満でＵＶ吸収が相対的に強いフルボ酸含有組成物 An algal growth promoter containing a fulvic acid-containing composition derived from brine (excluding brine itself) ,
The fulvic acid-containing composition has a molecular diameter of 0.0034 μm or less ,
Elemental analysis value is in the range of C (32 ± 10%), N (2.0 ± 10%), and the C / N is 15 or more,
And the growth promoter of algae which is the following (b) or (c) .
(B) A fulvic acid-containing composition having a molecular weight of 500 to 3,000 and having UV absorption
(C) A fulvic acid-containing composition having a molecular weight of 300 or more and less than 500 and relatively strong UV absorption
（ｂ）分子量５００〜３，０００でＵＶ吸収を持つフルボ酸含有組成物
（ｃ）分子量３００以上５００未満でＵＶ吸収が相対的に強いフルボ酸含有組成物 Containing fulvic acid in which boiled water is sonicated with ultrasonic cleaner and molecular diameter fractionated by at least two types of MF membrane and / or UF membrane is 0.0034 μm or less and (b) or (c) A method for growing algae, wherein the composition is added as a growth promoter to a culture solution containing algae.
(B) A fulvic acid-containing composition having a molecular weight of 500 to 3,000 and having UV absorption
(C) A fulvic acid-containing composition having a molecular weight of 300 or more and less than 500 and relatively strong UV absorption
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