复合固定化光合细菌及其处理养鱼水的效果

利用海藻酸钠和沸石,将含有球形红假单胞菌、荚膜红假单胞菌、沼泽红假单胞菌、万尼氏红微菌等菌体的复合光合细菌固定化,研究其对养鱼水的氮磷去除效果.比较了两种不同包埋材料固定化光合细菌处理养鱼废水的效果,对固定化光合细菌去除废水中氮磷的工艺条件进行了优化、并通过生物反应器连续处理养鱼水分析了处理后水质的效果.通过2 种固定化工艺的比较,确定了2 %沸石+2 %海藻酸钠(CA )的凝胶剂组合作为固定材料,其颗粒内生物活性最高.复合固定化光合细菌处理养鱼水的最佳条件为:厌氧光照条件下,颗粒粒径3 mm ,包埋比1 :5 ,颗粒投加量5 mg·L-1 ,4d 后养鱼水中NH4+-N 、PO43-和CODMn 的去除率分别为74.4%、84.26%、78.92%.此外,通过连续试验可以看出,固定化光合细菌具有明显的去除氨氮、磷酸盐的作用,其在净化养鱼水质方面具有非常明显的优越性.     

湖光微生物饲料添加剂研究：I.菌种来源及鉴定

湖光微五物饲料添加剂系采用我所从武汉东湖水体分离培养和筛选的光合细菌株制备的。我们对其中懈含的光合细菌菌株分类鉴定结果表明,它们分隶属于红假单胞菌属和红螺菌属,主要为沼泽红假单胞菌和深红红螺菌。并就有关分类问题作了讨论。     

湖光微生物饲料添加剂研究Ⅰ．菌种来源及鉴定

湖光微生物饲料添加剂系采用我所从武汉东湖水体分离培养和筛选的光合细菌菌株制备的。我们对其中包含的光合细菌菌株分类鉴定结果表明,它们分别隶属于红假单胞菌属（Ｒｋｏｄｏｐｓｅｕｄｏｍｏｎａｓ）和红螺菌属（Ｒｈｏｄｏｓｐｉｒｌｌｕｍ）,主要为沼泽红假单胞菌（Ｒｈｏｄｏｐｓｅｕｄｏ－ｍｏｎａｓｐａｌｕｓｔｒｉｓｖａｎＮｉｅｌ）和深红红螺菌（ＲｈｏｄｏｓｐｉｒｉｌｌｕｍｒｕｂｒｕｍＭｏｌｉｓｃｈ）。并就有关分类问题作了讨论。     

8株光合细菌的鉴定及其系统进化关系分析

目的为8株光合细菌(photosynthetic bacteria,PSB)作为益生菌株提供系统资料。方法用常规方法对8株PSB菌株的形态、培养特性及生理生化特征进行鉴定,同时定性分析菌株产生的类胡萝卜素和CoQ,测定菌株16S DNA序列并分析其系统进化关系,在GenBank中获取了8个16S DNA序列号。结果菌株鉴定结果表明:菌株2C、2c和13ing为沼泽红假单胞菌,Ga、Il106、WS8N为类球红细菌,MT1131为荚膜红细菌,rub为深红红螺菌。基于菌株16S DNA序列的系统进化树显示,同一种菌并不总是聚为一簇,但相隔较近;种属完全不同的菌株,尽管序列相似性高达97%以上,在系统进化树上相隔较远。结论8株PSB菌株的鉴定和系统进化关系分析结果为后续研究提供了背景资料,同时菌株在GenBank中获得的16S DNA序列号为菌株作上了生物标记,也为菌株的产权保护提供了依据。     

内蒙古碱湖中紫色非硫光合细菌的分离和鉴定

从内蒙古碱湖水样中分离得到一株紫色非硫光合细菌,命名为JH1-6.对该菌株进行了形态学观察、生理生化鉴定、活细胞吸收光谱以及16S rDNA序列分析.16S rDNA序列分析结果表明该菌株与沼泽红假单胞菌的16S rDNA序列同源性高达99%,结合形态特征和生理生化特性以及活细胞吸收光谱特征等,确定菌株JH1- 6在分类地位上属于沼泽红假单胞菌(Rhodopseudomonas palustris).     

光合细菌对小麦生长和光合功能的影响

【目的】探明光合细菌对小麦生长、产量及光合功能的影响。【方法】以尧麦16为材料,在不同生长时期施用光合细菌,研究光合细菌对小麦生长、产量及光合功能的影响。【结果】光合细菌培养液的不同成分可提高小麦旗叶SPAD值、光合速率及干物质积累。拔节期施用后,混合菌液对叶片SPAD含量促进作用最大,较不施用对照提高33.6%,小麦干物质积累较对照增加25.7%,单株籽粒重量增效为14.3%。单菌株实验处理中沼泽红假单胞菌促进作用最强,干物质积累和单株籽粒重量较培养基稀释液对照增效均为13.1%。不同施用时期的结果表明沼泽红假单胞菌对灌浆期和拔节期小麦促进效应最强,其中静息细胞可延长叶片功能期,使光合产物持续增加;无细胞培养液通过促进小麦营养生长,进而提高小麦产量。【结论】光合细菌可促进小麦生长,有效提高小麦生育过程中相关光合功能;施用时期应为小麦拔节期和灌浆期;光合细菌对小麦生长和产量促进作用是静息细胞和代谢活性物质综合作用的结果。     

一株能降解有机磷农药甲胺磷的光合细菌HP-1的分离及生物学特性的研究

从湖南农药厂甲胺磷废水和污泥中分离到光合细菌菌株18株,经过初步筛选得到1株可降解甲胺磷(metham idophos)的菌株HP-1,红色圆形菌落,边缘整齐光滑,中间稍稍突起,菌落直径0.64～2.60mm,菌革兰氏染色阴性G-,细菌单个细胞短杆形或弯曲杆形,(0.40～0.70)μm×(1.2～2.0)μm,不对称出芽分裂,细胞具有极生鞭毛或亚极生鞭毛,片层状光合内膜位于细胞膜下并与之平行,初步鉴定为沼泽红假单胞菌(Rhodopesudomonas plaustris).培养7d后,能够降解甲胺磷(65.20%,500m g/L和49.60%,1000m g/L),且在外加碳源时能提高其降解性能,还能降解乐果、毒死蜱、三唑磷和辛硫磷.该菌株发酵液能够促进萝卜种子萌发,提前发芽时间,具有促生生物学活性.     

沼泽红假单胞菌染色体的提取

近年来,由于工业产生的有机物对环境的污染越来越严重,推动了人们对能降解芳香物的沼泽红假单胞菌(Ｒ.ｐａｌｕｓｔｒｉｓ)的研究兴趣[1-8]。大多数芳香族化合物是严重危害人类健康的污染物质,为从基因水平研究沼泽红假单胞菌对芳香族化合物的生物降解机制,需要找到一种提取染色体的理想方法。泽沼红假单胞菌质膜构造比较特殊,且菌体富含蛋白质、色素、多糖等,给染色体提取造成一定难度。在本工作中采用了几种方法进行染色体的提取。1　材料与方法11　菌株来源:Ｙ6为本实验室分离、鉴定并保存的沼泽红假单胞菌。12　菌体培养121　沼泽红假单…     

铁和镍对光合细菌生长和产氢的影响

基于金属元素在生物体功能发挥中的作用以及它们参与光合细菌光合放氢的重要性,着重进行了铁和镍对沼泽红假单胞菌（Rhodopseudomonas palustris）Z菌株和一株红杆菌（Rhodobactersp.）细胞生长、光合放氢和光合色素合成影响的研究。结果表明,高浓度Fe3+可显著提高两菌株光放氢能力和生物合成能力,最适浓度的Fe3+可使其产氢能力分别达对照组的1.32倍和2.8倍,产氢得率分别为360.6mL/g和385.9 mL/g,生物量分别为对照组的1.42倍和1.54倍。9μmol/L Ni2+的添加可使两菌株产氢能力分别达对照组的1.48倍和1.96倍,产氢得率分别为429.7mL/g和456.3 mL/g。而当Ni2+浓度为12μmol/L时,两菌株的产氢活性受到不同程度的抑制,产氢得率分别降低46.7%和19.4%。在铁浓度相同时,添加6μmol/L Ni2+能明显促进两菌株的生长。而当Ni2+浓度大于6μmol/L时,细胞生长受到抑制。Fe3+和Ni2+对Rhodobactersp.菌株类胡萝卜色素有显著影响。研究结果显示, 426nm色素峰随铁浓度的增加和镍的添加而消失,同时,产氢活性提高。     

北桑寄生提取液对沼泽红假单胞菌生长的影响

目的研究北桑寄生提取液对沼泽红假单胞菌生长的影响。方法通过添加不同质量浓度的常规培养基和北桑寄生提取液,培养沼泽红假单胞菌,观察活菌量的变化规律、生长曲线及脱氢酶活性。结果在全量培养基和无常规培养基中,北桑寄生提取液浓度分别为(0~6.25)g/L和(0~12.50)g/L,有利于细菌生长。最佳转化和未转化条件下细菌的脱氢酶活性分别增加9.72和7.20倍,活菌数无明显变化。结论低质量浓度北桑寄生促进沼泽红假单胞菌生长,反之则表现为抑制作用。高浓度北桑寄生提取液可以增强脱氢酶活性。     

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38?°C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40?°C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.     

Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake

A recombinant photosynthetic bacterium, Rhodopseudomonas palustris, was constructed to simultaneously express mercury transport system and metallothionein for Hg(2+) removal from heavy metal wastewater. The effects of essential process parameters, including pH, ionic strength and presence of co-ions on Hg(2+) uptake were evaluated. The results showed that compared with wild type R. palustris, recombinant strain displayed stronger resistance to toxic Hg(2+), and its Hg(2+) binding capacity was enhanced threefolds. In the range of pH 4-10, recombinant R. palustris maintained effective accumulation of Hg(2+). The presence of 10 mg L(-1) Mg(2+), Ca(2+), Zn(2+) or Ni(2+) did not significantly influence Hg(2+) bioaccumulation by recombinant R. palustris from solutions containing 0.2 mg L(-1) Hg(2+), while Na(+) and Cd(2+) posed serious adverse effect on Hg(2+) uptake. Furthermore, EDTA treatment experiment confirmed that different from wild type R. palustris that mainly absorbed Hg(2+) on the cell surface, recombinant R. palustris transported most of the bound Hg(2+) into the cells.     

The morphological study of the cysts Pelomyxa palustris Greeff, 1874

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.     

Re-evaluation of osmotic effects as a general adaptative strategy for bacteria in sub-freezing conditions

We studied the molecular mechanisms of adaptation of a Siberian psychrophilic bacterial strain. Upon adaptation to 4 degreesC from 24 degreesC, the major changes observed were in the membrane and cell surface chemistry. There was no evidence for the formation of so-called 'compatible metabolites' that are thought to be responsible for the survival at very low to sub-freezing temperatures. The membrane fatty acids were shorter with an increased amount of unsaturation in the 4 degreesC cells compared to the 24 degreesC cells. The absence of a significant amount of phosphorylation in the membrane lipids at 4 degreesC compared to the levels at 24 degreesC was another significant difference. At 4 degreesC, the cell size was reduced in volume by a factor of approximately 14 compared to its size at 24 degreesC. The polar polysaccharide capsular layer was also significantly reduced. There were no significant changes in the protein profiles indicating that antifreeze proteins were not being produced. The results obtained here are consistent with observations and established theories and principles on and about the behavior of water in confined spaces. These indicate that ordering effects, surface charge and polarity are the key determinants of the freezing point and the type of ice structure that will be formed when water is confined to an area of the size of a bacterial cell.     

对沼泽红假单胞菌生产CoQ10添加前体物的优化

CoQ10因其具有多种生理生化功能而不仅用于药物也用作食品添加剂.本研究采用全因析中心复合设计及响应面法对茄尼醇、对羟基苯甲酸及甲硫氨酸三种CoQ10前体物的添加量进行了优化,以达到沼泽红假单胞菌J001最大量地生产CoQ10的目的.结果表明:经过对所得模型进行响应曲面法分析,当茄尼醇、对羟基苯甲酸及甲硫氨酸最佳添加量分别为124.8mg1-1,267.7mg1-1,130.2mg1-1时,得最大CoQ10产量预测值40.6[(mgCoQ10)(g干细胞)-1].对上述最佳组合进行试验验证得40.5±0.2[(mg CoQ10)(g干细胞)-1],很接近预测值,比对照(未加三种CoQ10前体物)CoQ10产量提高了109.8%.     

Ceramide-dependent caspase 3 activation is prevented by coenzyme Q from plasma membrane in serum-deprived cells

Coenzyme Q (CoQ) is the key factor for the activity of the eukaryotic plasma membrane electron transport chain. Consequently, CoQ is essential in the cellular response against redox changes affecting this membrane. Serum withdrawal induces a mild oxidative stress, which produces lipid peroxidation in membranes. In fact, apoptosis induced by serum withdrawal can be prevented by several antioxidants including CoQ. Also, CoQ can maintain cell growth in serum-limiting conditions, whereas plasma membrane redox system (PMRS) inhibitors such as capsaicin, which compete with CoQ, inhibit cell growth and induce apoptosis. To understand how plasma membrane CoQ prevents oxidative stress-induced apoptosis we have studied the induction of apoptosis by serum withdrawal in CEM cells and its modulation by CoQ. Serum-withdrawal activates neutral sphingomyelinase (N-SMase), ceramide release and caspase-3-related proteases. CoQ addition to serum-free cultures inhibited a 60% N-SMase activation, an 80% ceramide release, and a 50% caspase-3 activity induced by serum deprivation. Caspase activation dependent on ceramide release since C 2 -ceramide was only able to mimic this effect in 10% foetal calf serum cultured cells but not in serum-free cultures. Also, in vitro experiments demonstrated that C 2 -ceramide and ceramide-rich lipid extracts directly activated caspase-3. Taken together, our results indicate that CoQ protects plasma membrane components and controls stress-mediated lipid signals by its participation in the PMRS.     

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.     

Ion and oxygen fluxes in the unicellular alga Eremosphaera viridis

Plasma membrane fluxes of the large unicellular model algal cell Eremosphaera viridis (De Bary) were measured under various light regimes to explore the role of plasma membrane fluxes during photosynthesis and high light-induced chloroplast translocation. Plasma membrane fluxes were measured directly and non-invasively with self-referencing ion-selective (H(+), Ca(2+), K(+) and Cl(-)) potentiometric microelectrodes and oxygen amperometric microelectrodes. At light irradiances high enough to induce chloroplast migration from the cell periphery to its center, oxygen evolution declined to respiratory net O(2) uptake prior to any significant chloroplast translocation, while net K(+) and Cl(-) influx increased during the decline in photosynthetic activity (and the membrane potential depolarized). The results suggest that chloroplast translocation is not the cause of the cessation of O(2) evolution at high irradiance. Rather, the chloroplast translocation may play a protective role: shielding the centrally located nucleus from damaging light intensities. At both high and low light intensities (similar to ambient growth conditions), there was a strong inverse correlation between H(+) net fluxes and respiratory and photosynthetic net O(2) fluxes. A similar inverse relationship was also observed for Ca(2+) net fluxes, but only at higher light intensities. The net H(+) fluxes are small relative to the buffering capacity of the cell, but are clearly related to both photosynthetic and respiratory activity.     

Composition and localization of bacteriochlorophyll a intermediates in the purple photosynthetic bacterium Rhodopseudomonas sp. Rits

Rhodopseudomonas sp. Rits is a recently isolated new species of photosynthetic bacteria and found to accumulate a significantly high amount of bacteriochlorophyll (BChl) a intermediates possessing non-, di- and tetra-hydrogenated geranylgeranyl groups at the 17-propionate as well as normal phytylated BChl a (Mizoguchi T et al. (2006) FEBS Lett 580:137-143). A phylogenetic analysis showed that this bacterium was closely related to Rhodopseudomonas palustris. The strain Rits synthesizes light-harvesting complexes 2 and 4 (LH2/4), as peripheral antennas, as well as the reaction center and light-harvesting 1 core complex (RC-LH1 core). The amounts of these complexes were dependent upon the incident light intensities, which was also a typical behavior of Rhodopseudomonas palustris. HPLC analyses of extracted pigments indicated that all four BChls a were associated with the purified photosynthetic pigment-protein, as complexes described above. The results suggested that this bacterium could use these pigments as functional molecules within the LH2/4 and RC-LH1 core. Pigment compositional analyses in several purple photosynthetic bacteria showed that such BChl a intermediates were always detected and were more widely distributed than expected. Long chains in the propionate moiety of BChl a would be one of the important factors for assembly of LH systems in purple photosynthetic bacteria.     

Effect of Thiosulfate on the Photosynthetic Growth of Rhodopseudomonas palustris

Cell yields of Rhodopseudomonas palustris grown photoheterotrophically in pyruvate-mineral salts medium were increased by the photooxidation of added thiosulfate. However, thiosulfate had no effect on cell yields of cultures grown aerobically in darkness, although thiosulfate was also oxidized. The presence of thiosulfate increased photosynthetic cell yields on a variety of other organic substrates. Growth of cells in thiosulfate-containing medium, or the addition of thiosulfate to cells grown in thiosulfate-free medium, induced the formation of a thiosulfate-oxidizing system which quantitatively photooxidized thiosulfate to sulfate. R. palustris grew photoautotrophically with thiosulfate as an oxidizable substrate. Large amounts of supplemental bicarbonate carbon were incorporated when cells were grown photosynthetically in pyruvate-thiosulfate medium. Cells harvested after photoautotrophic or photoheterotrophic growth in fumarate-thiosulfate medium fixed (14)CO(2) at an 8- to 10-fold greater rate when provided with thiosulfate. The evolution of (14)CO(2) from pyruvate-1-(14)C during photoassimilation by R. palustris was greatly suppressed by the presence of thiosulfate. The increase in photoheterotrophic cell yields of R. palustris caused by the oxidation of thiosulfate may result from assimilation of substrate carbon which is normally evolved as carbon dioxide.