A New Species of Spirulina (Arthrospira) from the Mu Us Sandy Land of Inner Mongolia Autonomous Region in China

A new species, Spirulina (Arthrospira) bayannurensis B. Sh. Li et C. Qiao sp. nov. was firstly collected from the Lake of Bayannur, Mu Us Sandy Land, Inner Mongolia Autonomous Region in China on April 30, 1996 The morphological characteristics of the new species are described and illustrated by light micrographs, scanning and transmission electron micrographs. The type specimen of this new species is kept in Herbarium of Inner Mongolia Agricultural University.

极大节旋藻（Arthrospiramaxima）高分子量基因组文库构建及其应用

构建了极大节旋藻fosmid文库。该文库含有4300个克隆，重组率是100％，插入片段集中在30～36kh之间，平均为33kb，覆盖率为节旋藻基因组的25．8倍。随机挑选100个克隆进行双末端测序，得到110个序列，经生物信息学分析筛查到67条功能基因序列。选取了14对特异的末端序列作为序列标签位点并设计引物，采用STS-PCR反应池法通过多轮筛选，得到127个阳性克隆，按照相互位置关系，绘制了4个连续的克隆重叠群。该研究为深入了解极大节旋藻分子遗传特性和绘制物理图谱奠定了基础。

紫外线和^(60)Coγ射线对钝顶节旋藻(Arthrospira platensis)的诱变效应

将对数期的钝顶节旋藻(Arthrospira platensis)(原名钝顶螺旋藻,Spirulina platensis)A9菌株用超声波40 s预处理破碎成2个~4个细胞大小的片断,分别用不同剂量的紫外线(UV)和60Coγ射线处理,诱变后经3小时避光预培养分别接种于液体和固体培养基进行培养。固体培养时UV照射70 s和60Coγ照射3 500 Gy后无菌体存活,而在液体培养中高剂量处理的样品可以部分恢复生长。将诱变后的菌液分别加入20μg.mL-1的ρ-氟苯丙氨酸(-ρfluorophenylalan ine,FPA)和20μg.mL-1的刀豆氨酸(L-canavan ine su lphate,CS),放入光照培养箱中预培养3 d^4 d后涂于含相同浓度的氨基酸类似物FPA和CS平板,培养30 d后计算抗氨基酸类似物突变株的突变率。UV对A9菌株的完全致死剂量LD和存活率37%时的剂量D37值分别为70 s和22 s,LD/D37=3.18。A9菌株经UV诱变25 s时存活率为28.7%,抗FPA和抗CS突变率分别为2.31×10-3和1.50×10-3,最大诱变效应比(MME)分别为48.53和52.63。60Coγ射线对A9菌株的LD和D37值分别为3 500 Gy、1 250 Gy,LD/D37=2.8。60Coγ射线显著提高A9菌株的突变率,当诱变剂量为2 000 Gy、存活率为10.49%时A9菌株突变率最高,抗FPA和抗CS突变率分别为5.07×10-3和0.964×10-3,最大诱变效应比(MME)分别为241.43和74.15。60Coγ射线对钝顶节旋藻A9菌株的损伤比UV造成的损伤强烈(低的LD/D37值),比UV具有较大的诱变效应(高MME值)。采用两种诱变剂获得的抗FPA突变率都要高于抗CS突变率。通过诱变获得了大量的抗氨基酸类似物突变株,为遗传重组研究提供携带重要遗传标记的材料。

Isoenzymes and soluble protein in Arthrospira from alka-line lakes in Erdos Plateau, China and in exotic species

The authors compared isoenzymes of five enzymes and soluble protein in Arthrospira platensis (A3) and A. erdosensis (A4) from alkaline lakes in Erdos Plateau, Nei Monggo (Inner Mongalia), China and exotic species of A. platensis (A1) from Chad and A. maxima (A2) from Mexico by means of polyacrylamide gel electrophoresis. The results showed that the isoenzymes of EST, POD and soluble protein were polymorphic. Monomorphism and polymorphism were found in isoenzymes of AMY, CAT and SOD, and monomorphism was found only in the introduced species. The isoenzymes and soluble protein of the local species are all polymorphic. The number of bands in these species were in the order of A3>A4>A1>A2. A2 is the most primary, A1 from Chad Lake is relatively primary, A3 and A4 are ad- vanced species in evolution. Cluster analysis showed that the relation between the two introduced species are the closest to each other, and so too are the two local ones.

Effects of N source and nitrification pretreatment on growth of Arthrospira platensis in human urine

Culture ofArthrospiraplatensis （Spirulinaplatens） in human urine was investigated to get valuable biomass. NO3-N was the proper N source, in comparison with other N source, including urea, NH4-N and NO2-N. As a result, aerobic nitrification of human urine was performed, with above 93.6% nitrification percentage finally achieved with total-N （TN） load of 46.52 mg/（L.d）, in which Arthrospira platensis was successfully grown. The main compositions of the obtained biomass are close to those in Zarrouk medium. Thus, it is possible to culture Arthrospiraplatensis in nitrified human urine for food production within bioregenerative life support systems （BLSSs）.

Methodology of factorial design deriving guidelines for simulation of growth curve and production of sugars by Spirulina (Arthrospira) maxima

It is practical, economic and sometimes essential to derive rules or conclusions by performing lesser runs of experiments. In this part, a methodology based on 2 f factorial design was brought up to derive guidelines to simulate growth curve and production of sugars by Spirulina (Arthrospira) maxima . The growth curve or accumulation process of sugars was idealized by sets of straight lines limited by phase transfers of growth or accumulation of sugars. Normal analyses of the critical values of the transfers were used to derive their linear relationships with the initial conditions of the experimental factors. These linear functions were called guidelines and were used to simulate the growth curve or accumulation of sugars. Generalization of the guideline technique was determined by the kinetic limitation of nutrient nitrogen or sulfur that was dependent upon their stoichiometric deficiency directly derived from their initial values in the medium. This method uses the initial conditions of culture and does not need measurements of concentrations of nitrate, sulfate and pigments during cultivation. It is a practical and useful alternative way to trace and predict approximately the growth curve and production of sugars by S. maxima .

Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospira platensis in Escherichia coli

Arthrospira platensis is an economically important cyanobacterium; and it has been used widely in food and pharmaceutical industries. The phycocyanin(PC) from A. platensis is extremely valuable in medicine and molecular biology due to its antioxidation and anti-tumoring activity and applicability as fluorescence protein tag. In present study, two recombinant plasmids, one contained the phycocyanobilin(PCB)-producing genes(hox1 and pcyA) while the other contained the phycobiliprotein gene(cpcB) and the lyase gene(either cpcS/U or cpcT), were constructed and synchronically transferred into E. coli in order to test the the activities of relevant lyases for catalysing PCB addition to CpcB during synthesizing fluorescent PC holo-β-subunit(β-PC) of A. platensis. As was evidenced by the fluorescence emitted at a peak specific for PC, CpcB was successfully synthesized in E. coli, to which co-expressed PCBs attached though at a relatively low efficiency. The results showed that the attachment of PCBs to CpcB were carried out mainly by co-expressed CpcS/U but CpcB also showed some autocatalytic activity. Currently, no CpcT activity was detected in this E. coli expression system. Further studies will be conducted to improve the efficiency of fluorescent PC synthesis in E. coli.

Fractionation and characterization of polysaccharides from cyanobacterium Spirulina (Arthrospira) maxima in nitrogen-limited batch culture

By a sequentially selective extraction procedure, polysaccharides (PS) from cyanobacterium Spirulina (Arthrospira) maxima that can be bio functionally healthy components were fractionated in four parts including the culture medium (PSCM), the external layers of the cell (PSEL), the cell wall (PSCW) and storage granules (PSSG). The four fractionated parts of polysaccharides were characterized by gas liquid chromatography (GLC) equipped with FID and fused silica capillary column (15 m×0.53 mm i. d.). The contents of PSCM, PSEL, PSCW and PSSG were about 2.0%, 2.6%, 10.0% and 52.0% to cell dry matter, respectively. Glucose was almost the only monosaccharide in PSSG and PSCW and most predominant in PSEL, while in PSCM, xylose, rhamnose and glucose were the main ones. Two uronic acids represented by glucuronic acid and galacturoic acid, six neutral monosaccharides including fucose, rhamnose, xylose, mannose, galactose and glucose, and two possible unidentified sugars were found in PSEL and PSCM. These data are valuable for the selective productions of high added value sugars from Spirulina .

Bi-Enzymatic Conductometric Biosensor for Detection of Heavy Metal Ions and Pesticides in Water Samples Based on Enzymatic Inhibition in <i>Arthrospira platensis</i>

An original bi-enzymatic biosensor was designed by immobilizing Arthrospira platensis cells, called Spirulina, on gold interdigitated transducers. Phosphatase and esterase activities were inhibited, respectively, by heavy metals and by pesticides. Inhibition activities were observed with different mixtures of pesticides + heavy metals. The quantification limits for Cd2+ and Hg2+ are 10-20 M in mixture and in pure solution. The quantification limits of parathion-methyl, paraoxon-methyl and triazine are respectively 10-20 M, 10-18 M and 10-20 M in mixture and pure solutions. These results show that there is no synergistic effect between the two families of pollutants. Qualitative contamination of effluent samples and their purification after passing through a municipal wastewater treatment plant were observed by our bi-enzymatic biosensor and confirmed by classical analytical techniques for heavy metal ions.

Bioaccumulation of Pb^(2+) and Its Effects on Growth,Morphology and Pigment Contents of Spirulina (Arthrospira) platensis

A laboratory experiment was conducted to assess the bioaccumulation of Pb^2＋ and its effects on growth, morphology and pigment contents of Spirulina （Arthrosl）ira）platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations （0, 5, 10, 30, 50 and 10μgmL^-1）. The growth ofS. platensis was adversely affected by Pb〉 at high concentrations （30, 50 and 100μgmL^-1）. However, at low concentrations （5μgmL^-1）, Pb^2＋ could stimulate its growth slightly. The pigment contents （chlorophyllα and β carotene） were decreased in a dose-dependent manner. The highest reductions （67% and 53% respectively in chlorophyll α andβ carotene） were observed in 100 μg mL^-1 treatment group. The LC50 （96 h） of Pb^2- was measured as 75.34 μg mL^-1. Apart from a few cases of filament breakages at elevated concentrations （50 and 100 μgmL^-1）, morphological abnormalities are not specific. Metal bioaccumulation increased with Pb^2- concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mgg^-1 dry weight. The bioconcentration factor （BCF） reached to a peak at day 2, followed by a gradual reduction tbr all the exposure concentrations. S. platensis is able to tolerate considerably high Pb^2＋ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.

钝顶节旋藻(Arthrospira platensis)2-DE 分析蛋白制备方法改进及试用于螺旋手性差异蛋白研究

采用冻融破壁和蛋白质分步提取等技术,对钝顶节旋藻2-DE分析蛋白制备方法作了改进,并试用于螺旋手性差异表达蛋白研究,结果表明:(1)钝顶节旋藻细胞反复冻融5次完全破碎,用Tris-HCl提取液提取3次可提得近87%的水溶性蛋白,再用SDS(十二烷基磺酸钠)提取液可从沉淀中提得水不溶性蛋白;(2)将上述水溶性和水不溶性蛋白分别用TCA/丙酮法纯化后作双向电泳(2-DE)分析,可辨蛋白点分别达500和760多个,而两者间的匹配率仅7%,说明分离度好且质量高;(3)利用此法获得了13个与钝顶节旋藻螺旋手性相关的候选蛋白,其中5个为首次发现,候选蛋白功能涉及光合作用、能量代谢、物质外排和环境适应等;(4)本文所建的"冻融破壁分步提纯法"与现有节旋藻2-DE蛋白制备方法相比,具有操作简便、仪器设备简单、成本低廉及信息量更丰富等优点。

Cloning and analysis of [NiFe]-hydrogenase genes from Arthrospira platensis FACHB341

The complete [ NiFe]-hydrogenase genes were cloned from cyanobacterium, Arthrospira platensis FACHB341, using PCR and in-vitro cloning. The total length is 2078bp, including hoxY 549bp, hoxH 1431bp and the spacer in between 98bp. There is a stem-loop structure, downstream of the hoxY gene, serving as a transcription terminator. The deduced amino acid sequences of HoxY aud HoxH consist of acidic amino acid of 15.8% and 12.0%; alkalitropic amino acid of 11.9% and 15.3%; hydrophobic amino acid of 46.3% and 41.0%, respectively. The similarities of hoxY and hoxH genes in Arthrospira platensis FACHB341 to their homologues in other cyanobacteria were compared respectively. The secondary structures and 3D models of small and large subunits of [ NiFe]-hydrogenase were predicted by using 3D-PSSM.

极大节旋藻(Arthrospira maxima)生物钟基因kaiC的克隆及分析

以 kaiC 基因簇部分已知序列为引物设计位点,采用 PCR 反应池法从节旋藻基因组 fosmid 文库中筛选到 kaiC 基因克隆,通过步移测序获得了 kaiC 基因全长序列。kaiC基因编码区长1554 bp,基因 GC 碱基含量平均为41.76%,密码子第三位明显偏向于 U。在基因上游385 bp 范围内发现一个可能的启动子序列和一些基因调控元件。KaiC 蛋白分析发现了 Walker A、DXXG、不完整的 Walker B 等重要模体和具有催化作用的功能位点。Southern 杂交的结果证明 kaiC 基因在极大节旋藻中为单拷贝。极大节旋藻 kaiC 基因的特殊结构特征为研究 kai 基因簇的进化提供了重要的启示。

The Use of Cyanobacteria Arthrospira platensis and Cladoceran Daphnia magna as Complementary Protein and Lipid Sources in Transitional Diet for Common Carp (Cyprinus carpio L.) Nursery

This study was conducted to evaluate the use of cyanobacteria Arthrospira platensis and cladoceran Daphnia magna biomass as complementary protein and lipid sources in diet supplementation for common carp (Cyprinus carpio L.) nursery. Three experimental diets containing A. platensis and D. magna meal at different concentrations were compared to the commercial (control) diet. Each experimental diet (ED) was set to contain D. magna and A. platensis meal at a specific combination: 2% and 5%, 2% and 7%, and 4% and 5% for ED1, ED2 and ED3, respectively. The protein and lipid content of the experimental diets ranged from 43.20% to 44.60% dry weight (DW) and 10.64% to 13.42% DW, respectively;while the protein and lipid content of the control diet were 43.00% DW and 6.72% DW, respectively. After 20 days of feeding period, ED3 group obtained the highest final body weight (BW) (58.18 ± 35.24 mg), total biomass (1936 ± 1625 mg), food conversion rate (1.34 ± 0.04), and specific growth rate (12.86 ± 0.03% BW day–1) among all treatment groups (P > 0.05);while ED1 group obtained the highest survival (75.5% ± 7.47%) among all treatment groups (P > 0.05). The total bacterial count and total pathogenic Aeromonas sp. in the culture water of the treatment groups (ranged from 2.00 to 2.65 × 105 CFU mL–1 and 3.50 to 4.12 × 103 CFU mL–1, respectively) were lower compared to the water of the control group fed (3.73 × 105 and 4.70 × 103 CFU mL–1, respectively). No significant differences in physicochemical water quality parameters were observed among treatments (P > 0.05). The current study suggests that the combination of A. platensis and D. magna biomass can be used as complementary protein and lipid sources in diet supplementation for common carp larvae and can result in a comparable fish growth, survival and feed utilization in common carp culture at the nursery phase.

节旋藻(Arthrospira)3个样品镍铁氢化酶hoxY基因的克隆与测序

目的:为鄂尔多斯高原碱湖节旋藻的多样性研究提供基础资料和理论依据。方法:本文对鄂尔多斯高原碱湖钝顶节旋藻(Arthrospira platensis)、乍得湖钝顶节旋藻(A.platensis)及极大节旋藻(A.maxima)的镍铁氢化酶小亚基hoxY基因进行了克隆与测序,并进行同源性及亲缘关系的分析。结果:3个节旋藻样品hoxY基因部分序列长均为479 bp。鄂尔多斯高原碱湖钝顶节旋藻hoxY基因与不同来源的钝顶节旋藻的同源性高达99.5%,与极大节旋藻的同源性达99.9%。A.maxima与非洲乍得湖A.platensis间的亲缘关系更近,而鄂尔多斯高原碱湖钝顶节旋藻与乍得湖钝顶节旋藻的亲缘关系相对远。结论:hoxY基因保守性高,属内不同样品间序列变化很小。同种不同来源的钝顶节旋藻由于不同环境条件等因素的影响导致基因序列上碱基发生一定的差异。

采用基于GSS序列的极大节旋藻(Arthrospira maxima)的基因克隆策略及其应用实例

电子克隆是随着基因组计划的实施而发展起来的利用生物信息学手段进行基因克隆的新方法,已经公布的极大节旋藻GSS序列使得电子克隆极大节旋藻的功能基因成为可能。为建立极大节旋藻功能基因的高效克隆策略,应建立利用GSS序列克隆极大节旋藻功能基因的基本策略并加以应用。具体操作方法:对dbGSS数据库中的605条极大节旋藻GSS序列进行拼接及功能注释,并在分析编码区完整性的基础上设计引物并进行PCR扩增以获得基因全长序列,利用以上策略成功克隆了极大节旋藻功能基因ureaseβ和dUTPase的全长序列。

Biosynthesis of ^(13)C-Labeled Amino Acids and Sugars by Spirulina (Arthrospira) Maxima in a Parallelepiped Photobioreactor

This paper presents the investigation on biosynthesis of high-value-added amino acids and sugars labeled uniformly with stable isotope 13C by microalga Spirulina (Arthrospira) maxima in a parallelepiped photobioreactor. The kinetic data of both batch and continuous cultures with characterization of the amino acids and sugars are shown. The continuous culture without nutrients deficiency is for biosynthesis of amino acids, with tyrosine as one of the principal constituents, and the batch culture with deficiency in nitrogen is for biosynthesis of labeled glucose that is up to 64% versus dry mass of cells.

Accumulation of dysprosium,samarium,terbium,lanthanum,neodymium and ytterbium by Arthrospira platensis and their effects on biomass biochemical composition

The growth of Arthrospira platensis and physiological changes in biomass under the effects of six rare earth elements Dy,Sm,Tb,La,Nd and Yb were evaluated.Elements were tested by three concentrations of 10,20 and 30 mg/L.According to neutron activation analysis data A.platensis’s accumulation capacity toward studied elements changes in the following order of La>Dy>Nd>Sm>Yb>Tb.The results show that Dy and La ions stimulate biomass growth and Yb ions inhibit it,while Sm,Tb and Nd ions do not affect biomass accumulation.The contents of proteins and chlorophyll a are not affected by the presence of rare earth elements in the cultivation medium.Studied elements affect to different extents carbohydrates,phycobilins,β-carotene,lipids and MDA contents in spirulina biomass.Changes in the antioxidant activity under applied metal loads reveal a moderate stress in exposed A.platensis.Cyanobacterium A.platensis can be successfully used for bioremediation of natural water contaminated with REEs as well as REEs recovery from low polluted industrial effluents.

Raceway pond cultivation of a new Arthrospira sp.ZJWST-S1 in digested piggery wastewater treated by MBR and ozonation

A new Arthrospira strain named Arthrospira sp.ZJWST-S1 was isolated from a local digested piggery wastewater(DPW)storage pool in Jiaxing City,Zhejiang Province,China.It possessed good stain resistance against contaminants in DPW,which was pretreated with a membrane bioreactor(MBR).The strain was identified as Arthrospira platensis(A.platensis)based on its morphological characteristics and the 16S rDNA sequencing analysis.The effect of chrominance on growth of A.platensis ZJWST-S1 was investigated in a field raceway pond filled with MBR effluent or MBR effluent decolorized with ozonation.After ozonation,the chrominance of MBR effluent was decreased from 700 mg Pt/L to 150 mg Pt/L.Two runs of cultivation showed that A.platensis ZJWST-S1 grew faster in the ozone decolorized MBR effluent,the averaged biomass concentration being 0.907 g/L after 10 days of cultivation,close to that in a Zarrouk medium(0.969 g/L).By comparison,the biomass grew much slower in the non-decolorized MBR effluent(0.624 g/L).The pollutant removal was also benefited from the accelerative growth of A.platensis ZJWST-S1 in the decolorized MBR effluent.Almost all ammonium,61.2%of nitrate and 68.1%of phosphate were removed by the A.platensis ZJWST-S1 in the decolorized MBR effluent,much higher than the corresponding values of almost all ammonium,25.4%of nitrate and 36.5%of phosphate in the MBR effluent.Furthermore,the Arthrospira biomass harvested from the ozone decolorized MBR effluent after 10 d cultivation was with crude protein content of 59.1%±3.5%in dry algae powder.The content of Pb,As,Cd and Hg in biomass was also low enough to meet the Chinese Arthrospira Standard for Animal Feed(GB/T 17243-1998).This study showed that the new strain A.platensis ZJWST-S1 possessed potential to be used for producing animal feed and simultaneous removal of nitrogen and phosphorus in DPW.

纯培养节旋藻全基因组提取方法的比较研究

本研究从实验室保藏的节旋藻(Arthrospira)藻种出发,挑取形态不同的单藻丝体进行纯化培养,采用6种方法进行全基因组DNA提取的比较研究,而后以16S rRNA-ITS区基因作为分子标记对藻株进行相关序列测定和分子系统进化分析。结果表明,冻融CTAB法能够提取出包含染色体外DNA在内的节旋藻全基因组,高质量样品可以满足分子生物学实验要求;分子系统研究表明,纯化藻株皆为钝顶节旋藻,节旋藻与螺旋藻在分子鉴定中属间差异明显。