还原亚硒酸盐产生红色单质硒光合细菌菌株的筛选与鉴定

从实验室保藏的光合细菌中筛选出一株对亚硒酸钠还原效率较高的菌株S3,其亚硒酸钠还原产物通过透射电子显微镜及EDX(Electron-Dispersive X-ray)分析确定为红色单质硒。菌株S3的形态学特征、生理生化特征及光合色素扫描结果与固氮红细菌(Rhodobacter azotoformans)的特征基本一致;16S rDNA序列(GenBank登录号为DQ402051)在系统发育树中与固氮红细菌同属一个类群,序列同源性为99%。根据上述结果将菌株S3鉴定为固氮红细菌。初步研究了该菌株还原亚硒酸钠的特性,首次报道固氮红细菌具有还原亚硒酸盐产生红色单质硒的能力,为今后利用微生物方法治理环境中硒污染、利用微生物方法获得活性红色单质硒以及对微生物还原亚硒酸盐产生红色单质硒的机理研究奠定了良好的基础。     

微生物对硒的还原及其产物的应用研究进展——纪念硒发现200周年

硒是生命必需的微量元素,以硒代半胱氨酸(Sec,第21位氨基酸)和硒代甲硫氨酸(Se-Met)的形式加入到硒蛋白(酶)中。人畜硒摄入过量或不足均会导致很多疾病。微生物参与了Se(-Ⅱ)、Se(0)、Se(Ⅳ)和Se(Ⅵ)等各种价态间的转化。本文主要综述微生物对硒的还原及其生物学意义。微生物对硒的还原包括同化还原、异化还原以及在还原基础上进行的硒的甲基化。硒的同化还原主要是形成各种硒蛋白,满足微生物自身对硒的需求,食源性微生物对人畜补硒具有重要意义。高浓度硒酸盐和亚硒酸盐则可促使微生物进行异化还原并形成单质纳米硒颗粒。有的微生物会将还原态的Sec和Se-Met进一步转化为挥发态的甲基化硒。硒的异化还原和甲基化都是解毒机制,在硒污染环境的治理中具有重要意义。最后,阐述了单质纳米硒在医药、生物传感器和治理重金属污染等方面的应用前景,以及微生物合成CdSe荧光量子点的应用。     

一株假单胞菌(Pseudomonas alcaliphila MBR)好氧还原亚硒酸钠为红色单质硒

【目的】本实验室保藏的一株异化硝酸盐还原菌(Pseudomonas alcaliphila MBR),其能够在好氧环境下以有机碳源为电子供体,把易溶解、高毒性亚硒酸钠还原成为红色单质硒,本文对该菌株还原亚硒酸盐的特征进行了研究。【结果】结果表明该菌株可以在pH为6-11环境中生长,对亚硒酸钠有较强抗性,其MIC(minimal inhibitory concentration)可高达50 mmol/L。在5天时间内,菌体以柠檬酸钠为电子供体,把2 mmol/L亚硒酸钠完全还原为红色单质硒并主要积累于胞外。硝酸盐和还原型谷胱甘肽对菌体还原亚硒酸钠具有促进作用,初步确定菌体对亚硒酸钠的还原是细胞膜或细胞质中的某些物质催化的结果。【结论】本项研究为应用Pseudomonas alcaliphila MBR于生物反应器提供了重要基础。     

微生物硒代谢机制研究进展

硒(Se)是人与动物生命必需的微量元素,在医学保健和工业制造方面有着广泛的应用。硒在环境中有四种价态,包括硒酸盐Se O42-(+6)、亚硒酸盐Se O32-(+4)、单质硒Se0(0)和硒化物Se2-(-2)。微生物在硒的形态转化中扮演了重要的角色,影响着环境中硒的生物地球化学循环。本文主要从自然界中硒的循环以及微生物与硒代谢机制两个方面阐述微生物对硒的生物地球化学循环的重要性。     

硒代硫酸钠诱导HL60细胞凋亡——还原态硒的细胞毒性(英文)

细胞毒性研究认为Cd2+的释放是硒化镉(CdSe)纳米粒子的细胞毒性机制之一,而Se2-阴离子在纳米粒子中的毒性机制未知。作者研究了硒代硫酸钠(selenosulfate(SSeO3)2-)对HL60细胞的细胞毒性作用,发现10μmol/L的硒代硫酸钠可以显著抑制细胞活力,诱导细胞凋亡,出现了染色质凝聚、DNA ladder和G0/G1凋亡亚峰。线粒体膜电位显著降低的同时,促凋亡蛋白Bax的免疫荧光增加。结果表明还原态的Se2-阴离子有显著的细胞毒性作用,可以诱导HL60细胞凋亡。同时也暗示Se2-阴离子的释放可能是含Se2-纳米粒子(比如硒化镉的量子点)细胞毒性的机制之一。     

沼泽红假单胞菌对亚硒酸盐还原脱毒的研究

主要研究沼泽红假单胞菌对亚硒酸盐还原脱毒作用及其脱毒机理。通过单因子实验、正交试验, 对影响亚硒酸盐还原脱毒的因素进行研究, 得到沼泽红假单胞菌还原亚硒酸盐的最佳条件为: 亚硒酸钠添加量是25 mg/L, 培养的第5天接种接种量15% (质量比)。在该条件下, 对亚硒酸钠去除率可达98.2%。研究发现, 亚硒酸盐还原酶主要存在于细胞质, 分子量约为182 kD, 由4个亚基组成。通过透射电子显微镜观察, 菌体表面出现粒径在5 nm?200 nm之间的高电子密度颗粒, 初步表明亚硒酸盐在沼泽红假单胞菌体内被     

硒蛋白

硒(Se)已被确认为是一种生物微量元素,它能共价结合到生物大分子、尤其是蛋白质中。硒蛋白是某些细菌、鸟类、哺乳动物(可能也包含植物)的酶系统的基本成份。一、细菌硒蛋白最早被鉴定的细菌硒蛋白是依赖硒的甲酸脱氢酶,该酶催化无氧条件下HCOOH?H_2+CO_2。Pinsent(954)指出,E·Coli甲酸脱氢酶的表达需要硒。Lester和Demoss(1971)则     

耐高盐枯草芽孢杆菌XP合成球形纳米硒及其抑制草莓病原真菌生物活性

生物方法合成纳米材料具有低能耗、高安全性以及环境友好等优良特点,因而备受人们关注。利用细菌将硒酸盐或亚硒酸盐还原为单质硒,不仅可以降低硒毒性,而且还能获得价值更高的生物纳米材料。文中选用可耐受高盐环境胁迫的枯草芽孢杆菌亚种Bacillus subtilis subspecies stercoris strain XP构建生物模型,分别以LB液体培养基和亚硒酸钠为介质和底物 (电子受体),解析菌株XP合成纳米硒的基本规律。通过扫描电镜 (Scanning electron microscope,SEM) 观察、X射线能谱分析 (X-ray energy dispersive spectral analysis,EDAX)、X射线衍射 (X-ray diffraction,XRD) 分析、傅里叶红外变换光谱 (Fourier transform infrared spectroscopy,FTIR) 技术对合成的纳米硒进行物理化学表征分析,同时选用草莓枯萎、红叶、紫斑病病原真菌对其抗菌活性进行分析。结果表明,菌株XP介导合成的单质硒为球形纳米颗粒 (Selenium nanoparticles,SeNPs),其生成量与反应时间呈正相关 (0–48 h),且细胞形态未发生褶皱或破损等变化 (耐受力强);SeNPs为非晶态,粒径范围在135–165 nm,表面元素组成以Se为主,同时存在C、O、N、S等有机元素;颗粒表面包裹生物大分子物质,-OH、C=O、N-H、C-H等官能团与SeNPs稳定性和生物活性密切相关;高浓度纳米硒对枯萎、红叶、紫斑病病原真菌均有显著抑制活性 (P<0.05),其中对草莓红叶病与枯萎病病原真菌的抑制活性明显优于对紫斑病病原真菌的抑制活性。总而言之,菌株XP不仅耐受高盐胁迫能力强,同时还可介导合成生物SeNPs,其合成的纳米硒颗粒具有良好的稳定性和生物活性,在草莓病害防治以及绿色富硒草莓种植等领域具有潜在的应用价值。     

正交试验分析不同作物的产量、含硒量与土壤酸碱度、硒酸盐、亚硒酸盐含量的相关性

采用正交试验研究了碎米荠、韭菜、大豆、马铃薯的产量、含硒量与土壤酸碱度、硒酸盐、亚硒酸盐含量的关系。结果表明:影响作物含硒量最大的因素是作物品种,不同作物间差异极显著。在土壤中施用硒酸钠和亚硒酸钠均能够提高作物含硒量,用量均以1.0 mg/kg为宜;硒酸钠会使作物产量降低,用量过高使作物硒吸收总量下降;土壤pH值增加有利于植物对硒的吸收,但综合考虑作物产量、含硒量和硒摄入总量,土壤适合的pH值应在6.7～7.9之间。     

富硒螺旋藻培养技术研究

采用富硒技术对印项螺旋藻培养进行强化,对硒（IV）浓度和亚硫酸盐的影响,以及硒的生物富集及其对藻细胞分子官能团结构的影响等进行了较为详细的研究,并对相关的可能机理进行了讨论。研究发现,硒对印顶螺旋藻生长具有刺激或抑制的双重作用。在0.02mg/L-411.00mg/L浓度范围内,硒不仅可以加快印顶螺旋藻的生长,而且还可以提高其生物量;同时,钝顶螺旋藻对硒的事集随着硒浓度的增加而增加,较为缓慢的生长利于钝顶螺旋藻对硒的富集。研究还证实,NaSO3会减轻高浓度Na2SeO3对印顶螺旋藻生长的毒性,富硒培养不会对藻细胞分子官能团结构产生损害。实验得出钝顶螺旋藻富硒培养较佳的硒处理浓度在10mg／L－40mg／L。     

Reduction of Selenium Oxyanions in Wastewater Using Two Bacterial Strains

The biological reduction of selenium oxyanions is capable of reducing both selenate and selenite to insoluble elemental selenium. In this process, however, bacteria inevitably require expensive chemicals such as yeast extract in almost all cases. Therefore, the reduction of selenium oxyanions with inexpensive alcohol would be more practical. A Pseudomonas sp. strain 4C‐C isolated from a sludge in a wastewater treatment facility was able to reduce selenate to selenite using ethanol as an electron donor for its anaerobic respiration, but could not reduce selenite to elemental selenium. Paracoccus denitrificans JCM‐6892, on the other hand, was observed to be able to reduce selenite to elemental selenium in the presence of ethanol, but not selenate to selenite. Therefore, a mixture containing a suspension of Pseudomonas sp. strain 4C‐C and P. denitrificans JCM‐6892 cells allowed selenate to be reduced to insoluble elemental selenium via selenite in the presence of ethanol and was also capable of reducing nitrate to nitrogen gas. Aiming at simplicity of the recovery process of insoluble elemental selenium, a polymeric gel immobilized mixture of the two bacterial strains was examined using ethanol as an electron donor. The immobilized mixture could therefore reduce not only selenate to elemental selenium, but also nitrate to nitrogen gas in a single step. The gel that immobilized the microbial mixture changed its color during the process to bright red and no red elemental selenium was left in the wastewater. This indicates that the reduced elemental selenium was completely absorbed in the gel. This simple bacterial combination would therefore be effective in the presence of ethanol to reduce selenium oxyanions in various wastewaters containing selenium and the other oxyanions.     

Laboratory-scale continuous reactor for soluble selenium removal using selenate-reducing bacterium,Bacillus sp. SF-1

A model continuous flow bioreactor (volume 0.5 L) was constructed for removing toxic soluble selenium (selenate/selenite) of high concentrations using a selenate-reducing bacterium, Bacillus sp. SF-1, which transforms selenate into elemental selenium via selenite for anaerobic respiration. Model wastewater contained 41.8 mg-Se/L selenate and excess lactate as the carbon and energy source; the bioreactor was operated as an anoxic, completely mixed chemostat with cell retention time between 2.2-95.2 h. At short cell retention times selenate was removed by the bioreactor, but accumulation of selenite was observed. At long cell retention times soluble selenium, both selenate and selenite, was successfully reduced into nontoxic elemental selenium. A simple mathematical model is proposed to evaluate Se reduction ability of strain SF-1. First-order kinetic constants for selenate and selenite reduction were estimated to be 2.9 x 10(-11) L/cells/h and 5.5 x 10(-13) L/cells/h, respectively. The yield of the bacterial cells by selenate reduction was estimated to be 2.2 x 10(9) cells/mg-Se.     

An <Emphasis Type="Italic">Azospira oryzae</Emphasis> (syn <Emphasis Type="Italic">Dechlorosoma suillum</Emphasis>) Strain That Reduces Selenate and Selenite to Elemental Red Selenium

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se⁰) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se⁰ under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal eˉ donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 ± 0.04 μm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.     

Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria, Sulfurospirillum barnesii, Bacillus selenitireducens, and Selenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, approximately 300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H(2)Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.     

Reduction of selenium oxyanions by unicellular,polymorphic and filamentous fungi: Cellular location of reduced selenium and implications for tolerance

Summary The ability of several filamentous, polymorphic and unicellular fungi to reduce selenite to elemental selenium on solid medium was examined.Fusarium sp. andTrichoderma reeii were the only filamentous fungi, of those tested, which reduced selenite to elemental selenium on Czapek-Dox agar resulting in a red colouration of colonies. Other organisms (Aspergillus niger, Coriolus versicolor, Mucor SK, andRhizopus arrhizus) were able to reduce selenite only on malt extract agar. Several fungi were able to grow in the presence of sodium selenite but were apparently unable to reduce selenite to elemental selenium, indicating that other mechanisms of selenite tolerance were employed, such as reduced uptake and/or biomethylation to less toxic, volatile derivatives. Sodium selenate was more toxic toFusarium sp. than selenite, and the toxicity of both oxyanions was increased in sulphur-free medium, with this effect being more marked for selenate. Scanning electron microscopy ofAspergillus funiculosus andFusarium sp. incubated with sodium selenite showed the presence of needle-like crystals of elemental selenium on the surfaces of hyphae and conidia, while transmission electron microscopy ofA. funiculosus revealed the deposition of electron-dense granules in vacuoles of selenite-treated fungi. Several yeasts were able to grow on MYGP agar containing sodium selenate or sodium selenite at millimolar concentrations. Sone, notablyRhodotorula rubra andCandida lipolytica, and the polymorphic fungusAureobasidium pullulans were also effective at reducing selenite to elemental selenium, resulting in red-coloured colonies.Schizosaccharomyces pombe was able to grow at selenite concentrations up to 5 mmol L^–1 without any evidence of reduction, again indicating the operation of other tolerance mechanisms.     

Selenium metabolism in Escherichia coli

Escherichia coli will reduce selenite (SeO 3 2- ) andselenate (SeO 4 2- ) to elemental selenium Se 0 . Seleniumwill also become incorporated intoproteins as part of the amino acids selenocysteine or selenomethionine.The reaction of selenitewith glutathione produces selenodiglutathione (GS-Se-GS). Selenodiglutathioneand itssubsequent reduction to glutathioselenol (GS-SeH) are likely the key intermediatesin the possiblemetabolic fates of selenium. This review presents the possible pathwaysinvolving selenium in E. coli. Identification of intermediates and potentialprocesses from uptake of the toxic oxyanions through to theirdetoxification will assist us inunderstanding the complexities of metalloid oxyanion metabolism in thesebacteria.     

Rhizosphere-induced selenium precipitation for possible applications in phytoremediation of se polluted effluents

Two bacterial isolates were obtained in axenic culture from the rhizosphere soil of Astragalus bisulcatus, a legume able to hyperaccumulate selenium. Both strains resulted of particular interest for their high resistance to the toxic oxyanion SeO3(2-) (selenite, Se(IV)). On the basis of molecular and biochemical analyses, these two isolates were attributed to the species Bacillus mycoides and Stenotrophomonas maltophilia, respectively. Their capability in axenic culture to precipitate the soluble, bioavailable and highly toxic selenium form selenite to insoluble and relatively non-toxic Se(0) (elemental selenium) was evaluated in defined medium added with 0.2 or 0.5 mM Se(IV). Both strains showed to completely reduce 0.2 mM selenite in 120 h, while 0.5 mM Se(IV) was reduced up to 67% of the initial concentration by B. mycoides and to about 50% by S. maltophilia in 48 h. Together in a dual consortium, B. mycoides and S. maltophilia increased the kinetics of selenite reduction, thus improving the efficiency of the process. A model system for selenium rhizofiltration based on plant-rhizobacteria interactions has been proposed.     

Accumulation of selenium in a model freshwater microbial food web.

The transfer of selenium between bacteria and the ciliated protozoan, Paramecium putrinum, was examined in laboratory cultures. The population growth of the ciliate was not inhibited in the presence of the highest concentrations of dissolved selenite or selenate tested (10(3) micrograms liter-1). Experiments with radioactive 75selenite or 75selenate indicated that accumulation of selenium by ciliates through time was low when feeding and metabolism were reduced by incubating at 0 degrees C. However, selenium accumulated in ciliate biomass during incubation with dissolved 75Se and bacteria at 24 degrees C and also when bacteria prelabeled with 75Se were offered as food in the absence of dissolved selenium. When 75Se-labeled bacterial food was diluted by the addition of nonradioactive bacteria, the amount of selenite and selenate in ciliates decreased over time, indicating depuration by the ciliates. In longer-term (> 5-day) fed-batch incubations with 75selenite-labeled bacteria, the selenium concentration in ciliates equilibrated at approximately 1.4 micrograms of Se g (dry weight)-1. The selenium content of ciliates was similar to that of their bacterial food on a dry-weight basis. These data indicate that selenium uptake by this ciliate occurred primarily during feeding and that biomagnification of selenium did not occur in this simple food chain.