Development of Effective Biological Treatment Process for Acid Mine Drainage

Acid mine drainage (AMD) is acidic and generally enriched with iron, aluminum, sulfate and heavy metals, such as lead and cadmium. AMD is a growing problem of emerging concern that cause detrimental effects to the environment and living organisms. Yanahara mine in Misaki Town, Okayama, Japan, had mainly produced pyrite for sulfuric acid manufacture. Although it was closed in 1991, AMD is being generated from the mine now. A passive treatment based on the biological oxidation of ferrous iron is a promising strategy for AMD remediation. AMD from Yanahara mine is treated in a plant using iron-oxidizing bacteria, Acidithiobacillus ferrooxidans and Ferrovum spp. The AMD generation continues for several centuries with dramatic consequences on the receiving environments. Therefore, the development of sustainable and cost effective treatment process is required. A development of the effective biological treatment process with an iron oxidation reactor operated at pH 3.5 is described in this report. Economic aspects are also discussed

硫黄で増殖したAcidthiobacillus caldus GO-1株による還元型硫黄化合物の酸化機構

The oxidation of reduced sulfur compounds was studied by using resting cells of sulfur-grown Acidithiobacillus caldus strain GO-1. The optimum pHs for the oxidation of thiosulfate, tetrathionate, sulfur, sulfite and sulfide were 2, 3, 3-6, 7 and 7, respectively. The highest oxidation rate was observed with sulfite. The oxidation rates of the reduced sulfur compounds were measured in the absence or presence of inhibitors and uncouplers. 2, 4-dinitrophenol (DNP) and carbonyl cyanide-m-chlorophenylhydrazone (CCCP) strongly inhibited the oxidations of sulfur and sulfite. N-Ethylmaleimide (NEM) strongly inhibited the oxidation of tetrathionate and sulfur. 2-heptyl-4-hydroxy-quinoline-N-oxide(HQNO) inhibited the oxidation of sulfur and sulfite. The results suggested that tetrathionate was oxidized in the periplasmic space, and sulfur and sulfite were oxidized in the cytoplasm. Pyridine ferrohemochromes prepared from the membrane of strain GO-1 cell revealed the involvement of cytochromes b and c. Ubiquinol oxidase activity was detected in strain GO-1 cell, but cytochrome c oxidase measured by using mammalian cytochrome c as an electron donor was not detected in the cell. On the basis of the results a model for the metabolism of the reduced sulfur compounds by At. caldus strain GO-1 was proposed.硫黄で増殖したAcidithiobacilluscaldusGO-1株の休止菌体を用いて，還元型硫黄化合物の酸化機構を検討した．GO-１株は，チオ硫酸，テトラチオン酸，元素硫黄，亜硫酸及び硫化物の酸化活性を持っており，その最適pHは，それぞれ２，３，３-６，７及び７であった．これらの酸化活性の中で，亜硫酸の酸化速度が最も高かった．阻害剤やアンカップラーを用いて，還元型硫黄化合物の酸化への影響を調べた結果，DNPとCCCPによる元素硫黄及び亜硫酸酸化活性の強い阻害，NEMによるテトラチオン酸及び硫黄酸化活性の強い阻害，およびHQNOによる元素硫黄及び亜硫酸酸化活性の阻害が観察された．これらの結果は，テトラチオン酸がペリプラズマで，硫黄及び亜流酸が細胞質で，それぞれ酸化されることを示唆した．細胞膜からピリジンフェロヘモクロームを調製して，シトクロームの構成成分を検討した結果，ヘムb及びcが検出された．また，末端酸化酵素の活性を検討した結果，ユビキノール酸化酵素の活性は検出されたが，哺乳類のシトクロームを用いて測定したシトクロームc酸化酵素の活性は検出されなかった．これらの結果に基づいて，At.caldusGO-１株の還元型硫黄化合物の酸化機構を考察した

Study on the Mechanism on Bacterial Adaptation to Deep-Sea Environment

The world's oceans cover 70% of the eath's surface,with about 3,800m of average depth.Altough　the deep-sea environment with its high pressure and low temperatures is too extreme for most terrestrial and marine surface microorganisms,many barotolerant and barophilic bacteria have been found inhabiting the deep-sea.It is exyremely important for barophilic or barotolerand deep-sea bacteria to maintain the physiological functions of cytoplasmic membrane,which serves many vital functions.The fluidity of this cytoplasimic membrane composed of phospholipids and poteins is essential for the physiological functions of cells.As higher hydorstatic pressure raise the melting point of lipids and cause phase transition of lipid under pressurs of up to 100MPa,barotolerant and barophilic bacrteria under high hydostatic pressure appear to regulate the composition of their membrane phospholipids. Therfore the characrization of cytoplasmic membrane under high pressure is indispensable to clarify the mecanisms of bacteria adaptation to the deep-sea enviroment.The effects of pressure and temperature acid compositon of barotolerant deep-sea bacteria were investigated.Deep-sea bacteria maintained their membrane fluidity by increasing the content of unique fatty acid in phospholipids under high hydrostatic pressure.Gene expression seems to be necessary for the synthesis of unique fatty under high hydrostatic pressure.細菌は、外界の刺激に応答する機構を備えており、既に温度変化、浸透圧変化、放射線などの物理的刺激に応答した遺伝子発現調節機構が分子レベルで明らかにされている。深海から分離した耐圧性菌の耐圧機構を明らかにするため、細胞膜の脂肪酸組成と圧力の関係を検討した結果、加圧下で特異的な不飽和脂肪酸を増加させていることが明らかとなった。また、加圧下で培養した耐圧性細菌の細胞膜の酵素活性が大気圧下で培養したものよりも耐圧性になっていることを明らかにした。筆者らの単離した耐圧性菌の高圧下での特異的な脂肪酸の増加は、RNA合成阻害剤によって阻害されることから、細胞が圧力という刺激を感知し、その情報によって特異的な遺伝子の発現が引き起こされているものと推測された。この応答機構の分子的レベルでの解析によって、細菌の高圧環境への適応機構を明らかにする糸口が得られることが期待される。高圧環境下で細菌が生息するためには、細胞膜の機能だけでなくさまざまな酵素タンパク質や高分子合成系等の生化学的機能が、高圧環境下でも維持されていることが必要である。各種酵素タンパク質やタンパク質合成系の耐圧機構に関しては十分な生化学的解析がなされているとは言い難く、今後の研究課題として残されている。著者らは、加圧下での驚異的な脂肪酸の増加に、圧力に関連した遺伝子の発現が関与していることを示唆したが、既に加圧下で特異的に発現する遺伝子の存在が報告され、加圧下で特異的に発現する遺伝子が分離されている。また、圧力によって制御されるプロモーター部位も同定されている。圧力は、我々陸上環境に適応した生物にはなじみづらいが、海洋では極めて普遍的な物理的因子であり、その物理的因子を細菌がどのように認識し適応しているかを分子レベルで知ることは、海洋微生物資源の有効利用を図るときに非常に重要であると考えられる。今後、耐圧性細菌や好圧性細菌の加圧下における生理・生化学的な研究の進展によって、耐圧機構が分子レベルで明らかにされることが期待される

Electronic Phase Transition of Cesium Metal under High Pressure

The isostructural phase transition of cesium metal, CsII←→CsIII, under pressure is discussed in terms of the crystalline energy as a function of volume. Using the augmented plane wave (APW) method and the quantum defect method, the energy bands are calculated at symmetry points in the Brillouin zone for the ran e of relative volume V/V_0 (V_0 : the volume at normal pressure) from 1.30 to 0.24. As the volume decreases, there appears a pronounced tendency of d-states to fall lower relative to s-states, which rise significantly at V/V_0≤0.6. Besides overall rise of the band energy with decreasing volume, a small humped portion is found at 0.4≤V/V_0≤0.5, this being attributed to the shift of electrons into the vicinity of X. The transition beyond this portion is accompanied by a significant volume change. Thus the CsII←→CsIII transition is interpreted as an electronic one. A calculation using the Green\u27s function method is also carried out and the results are compared with those by the APW method

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

AbstractWe have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level

On the Magnetocrystalline Anisotropy of Iron Selenide Fe_7Se_8

Magnetization measurements along the α- and c-axes of a single crystal of a compound Fe_7Se_8 have been carried out in magnetic fields up to 92 kOe over the temperature range from 4.2° to 300°K, together with magnetic torque measurements on the bc-plane up to 9 kOe. The results of magnetization measurements along the c-axis lead to the magnetocrystalline anisotropy energy of the form, E(θ)=-K_0|cosθ|+K_3 cos^2θ+K_4 cos^4θ, the last term being for minor correction. In this expression θ is an angle between magnetization and field direction. The measured values of anisotropy constants K_0 and K_3 at 290°, 77° and 4.2°K are K_0=0.3×10^6, 32×10^6 and 65×10^6 erg/cc and K_3=2.5×10^6, 18×10^6 and 35×10^6 erg/cc, respectively. From the magnetocrystalline energy mentioned above, it can be shown that the magnetic moment takes a conical distribution around the c-axis at low temperatures. The results of torque measurements on the bc-plane is reproduced by assuming a triad crystal domain structure

自然環境より単離したThibacilus ferroxidansの鉄酸化酵素及び硫化水素酸化酵素活性

It has been reported that both iron oxidase and hydrogen sulfide: ferric ion oxidoreductase (SFORase) were involved in bacterial leaching of metal ions from sulfide ores, and the amount of Cu2+ solubilized from copper ore by iron-oxidizing bacterium differed from strain. The activities of iron oxidase SFORase of iron-oxidizing bacteria isolated from the natural environments were determined. Iron-oxidizing activity and SFORase activity of 200 strains ranged from 1.20-1.65γmol/mg/min and from 0.11-2.80 γmol/mg/min, respectively. The findings that a remarkable difference was observed in the levels of SFORase activity, but not in iron-oxidizing activity, suggest that SFORase, but not iron oxidase, is the enzyme that determines the bacterial leaching activity of this bacterium.鉄酸化酵素と硫化水素酸化酵素の両方が硫化鉱石からの金属イオンのバクテリアリーチングに関与していること、銅鉱石から溶出する銅イオンの量が鉄酸化細菌の菌株間で異なることが知られている。鉄酸化酵素及び硫化水素酸化酵素活性が自然環境から単離した鉄酸化細菌に対して決定された。200株の鉄酸化細菌の鉄酸化酵素及び硫化水素酸化酵素活性は、それぞれ、1.20-1.65γmol/mg/min及び0.11-2.80γmol/mg/minの範囲にあった。これら菌株間において、鉄酸化酵素ではなく硫化水素酸化酵素活性に大きく違いがあるという発見は、前者ではなく後者がこの細菌のバクテリアリーチング活性を決定する酵素であることを示唆している

Effects of Murphy number on quadrupedal running gait based on a simple model

The 11th International Symposium on Adaptive Motion of Animals and Machines. Kobe University, Japan. 2023-06-06/09. Adaptive Motion of Animals and Machines Organizing Committee.Poster Session P4

16S-23S rDNA スペーサー領域の制限断片長多型解析によるAcidithiobacillus 属3種、A.ferroxidans、A.thiooxidans およびA.caldus の識別

The PCR-amplified 16S-23S rDNA intergenic spacer regions (ISRs) of Acidithiobacillus ferrooxidans, A. thiooxidans, and A. caldus strains were scquenced and evaluated for differentiation and identification of these bacteria. The total length of the 16S-23S ISRs of A. ferrooxidans and A. thiooxidans strains and A.caldus GO-1 were 441, 456, nd 379bp, respectively. Two genes. encoding tRNA and tRNA, and the box A-like sequences were highly conserved in the ISRs of all Acidithiobacillus species. The restriction fragment length polymorphism (RFLP) profiles of the PCR-amplified 16S-23S rDNA ISRs digested by HaeIII and AluI could clearly discriminate A. ferrooxidans from A. thiooxidans and A. caldus. The results indicated that RFLP analysis of the 16S-23S ISRs is an easy and rapid method for discrimination and identification of Acidithiobacillus species.Acidithiobacillus ferrooxidans，A.thiooxidansおよびA.caldusの16S-23S rDNAスペーサー領域の塩基配列を決定し，これらのバクテリアの識別・同定への有効性を評価した．A.ferrooxidans，A.thiooxidansおよびA.caldusのスペーサー領域の長さは，それぞれ441，456および379bpであった．３種のAcidithiobacillusスペーサー領域では，tRNA，tRNAをコードする遺伝子の塩基配列が高度に保存されていた．PCR増幅した16S-23S rDNAスペーサー領域をHaeおよびAluで酵素処理することによって得られた断片の解析によって，A.ferrooxidansをA.thiooxidansとA.caldusから識別できた．Acidithiobacillus種の16S-23S rDNAスペーサー領域の制限断片長多型解析は，Acidithiobacillus属の種の同定およびA.ferrooxidansに属する株の同定のための迅速で，技術的に簡便な方法であることが明らかとなった

好酸性鉄酸化細菌 Acidithiobacillus ferrooxidans の染色体凝集・分配タンパク質（ScpB）の性質

　Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide. This bacterium uses ferrous iron and reduced inorganic sulfur compounds (RISCs) as energy sources. Transcriptions of genes thought to be involved in the oxidation of RISCs have been known to be highly activated in A. ferrooxidans cells grown on RISCs, while transcriptions of genes involved in the iron oxidation were repressed in the cells grown on RISCs. A gene encoding a putative chromosome segregation and condensation protein (ScpB) with a helix-turn-helix motif was found in the upstream region of sulfide : quinone oxidoreductase gene, whose expression was up-regulated in cells grown in sulfur and tetrathionate. A semi-quantitative PCR analysis using cDNA prepared from iron-, sulfur-, or tetrathionate-grown cells revealed that the transcription of scpB was up-regulated in cells grown on sulfur or tetrathionate as the energy source. Electrophoretic mobility shift assays were employed to examine whether the ScpB functions as a transcription factor. The result indicated that the recombinant His-tagged ScpB protein was able to nonspecifically bind in vitro to DNA. This is the first report on a direct association of ScpB with DNA.　Acidithiobacillus ferrooxidans は，低品位の銅鉱石から銅を回収するバイオリーチングにおいて使用される微生物 の一つである．この細菌は，エネルギー源として二価鉄イオンや還元型無機硫黄化合物（RISC）を使用する．鉄の酸化に関与する遺伝子の転写は，A. ferrooxidans が RISC で生育したときには抑制されるが，RISC の酸化に関与すると考えられている遺伝子の転写は活性化されることが知られている．硫黄やテトラチオン酸で生育したときにその発現が上方制御される硫化水素：キノン酸化還元酵素のすぐ上流に，ヘリックスターンへリックスモティーフを持つ，ScpB と推定されるタンパク質をコードする遺伝子が存在していた．鉄，硫黄，テトラチオン酸生育細胞から調製した cDNA を用いた半定量的 PCR 分析の結果，硫黄やテトラチオン酸で生育した細胞内の scpB 遺伝子の転写は，鉄生育細胞と比較すると上方制御されていた．組換え ScpB タンパク質を用いたゲルシフトアッセイ法で，ScpB が転写制御因子として機能するかどうかを調べた．その結果，ScpB は DNA に結合したが，結合の特異性はなかった．ScpB が直接 DNA と相互作用する報告はこれまでになかった