紫球藻的培养与利用（综述）

紫球藻为单细胞红藻,其细胞富含红藻多糖、多不饱和脂肪酸(主要为AA和EPA）及藻胆蛋白等高价值生物活性物质,在医药和精细化工领域有广泛的应用前景。本文综述紫球藻的生物学特性、生物活性物质、培养与培养特性等方面的研究概况。     

有机物质对紫球藻生长的影响

研究了有机碳、氮源扩B族维生素对紫球藻生长的影响。葡萄糖促生长作用最佳,添加2％（W／V）葡萄糖时,藻细胞生长速度比对照组明显提高,培养10d收获的生物量增加92.6％,培养液中的溶解氧含量和藻体叶绿素a含量也有变化,有机氮源的利用率低,仅蛋白胨、酵母汁可被利用。维生素B2和B12也有促长作用。     

平板式光生物反应器中紫球藻培养条件的优化

研究了平板式光生物反应器中紫球藻(Porphyridium cruentumNaegeli)的培养条件,运用均匀设计法对光照强度、通气速率、装液量、接种密度以及pH等影响紫球藻生长的因素进行优化,获得了在平板式光生物反应器中培养紫球藻的最佳条件:光照强度10 000 lx、通气速率350 L.h-1、装液量6 L、藻细胞接种密度1.1×106mL-1、pH9.0。在最佳条件下藻体的生物量产率和生物量产量分别达到0.431 g.L-1.d-1和3.240 g.L-1,最大生长速率达0.652 g.L-1.d-1,胞外多糖含量高达0.665 g.L-1。另外,在培养过程中隔天补充培养液有利于紫球藻生物量的增加和胞外多糖的产生。     

三种紫球藻亲缘关系RAPD分析

利用RAPD技术对铜绿紫球藻（Porphyridium aerugineum 755）,淡色紫球藻（Porphyridium purpureum 806）和紫球藻（Porphyridium cruentum）的亲缘关系进行分析。从50个随机引物中筛选出25个种间多态性较强,重复性较好的引物。检测到233个位点。在233个条带中有186个多态性位点。多态位点比率为79.73%,平均每个引物扩增9个条带,多态性条带7个。聚类分析结果表明：铜绿紫球藻、淡色紫球藻和紫球藻之间的平均遗传距离为0.455,其中铜绿紫球藻和淡色紫球藻之间的遗传距离为0.413,铜绿紫球藻和紫球藻之间的遗传距离为0.556,淡色紫球藻和紫球藻之间的遗传距离最小为0.396。所以由RAPD试验的分析结果可以得出:三种紫球藻为独立的种, 其中淡色紫球藻和紫球藻是亲缘关系较近的两个不同种。紫球藻属种间个体DNA多态性比较丰富,因此利用RAPD技术可以从DNA水平上检测紫球藻属种间差异。     

紫球藻多糖化学结构解析

为明确紫球藻多糖的化学结构,本文采用化学分析和光谱分析方法对紫球藻多糖的一级糖链结构进行了分析。GC分析表明该多糖由木糖、葡萄糖和半乳糖组成,为一种杂多糖,其摩尔比为:2.96∶1.25∶3.06;红外光谱分析结果显示紫球藻多糖为硫酸化多糖,糖苷键类型为β构型;化学分析结果推断紫球藻多糖糖链连接方式以1→3为主,存在少量1→2,1→4,1→6键型,且半乳糖在支链或主链末端有较大量的存在,木糖和葡萄糖在主链或靠近主链区域有特定分布;NMR分析显示紫球藻多糖的硫酸酯基连在C-6上,且多糖的糖苷键为β型;GC-MS联机分析进一步确定紫球藻多糖为一种主要含有1→3糖苷键,并含有1→4,1→6糖苷键的杂多糖。综合上述分析,推断出紫球藻多糖的糖链主链的重复单元结构。     

绿光条件下氮浓度对紫球藻生长和生物活性产物合成的影响

本文以紫球藻FJ-12为藻株,在发光二极管(LED)绿光条件下,研究不同氮浓度(0、0.5、1和1.5 g/L)对紫球藻细胞的生长和生物活性产物(藻胆蛋白、胞外硫酸酯多糖和多不饱和脂肪酸)合成的影响.结果 表明:在氮浓度为1 g/L的条件下,紫球藻可获得最大生物量,为3.09 g/L.藻细胞在氮浓度为1 g/L和1.5...     

紫球藻胞外多糖抗呼吸道合胞病毒(RSV)活性研究

采用体外细胞培养的方法,在Hela细胞系上检测了来自紫球藻的胞外多糖及其组分的抗呼吸道病毒(RSV)活性。发现紫球藻胞外多糖对呼吸道合胞病毒具有强烈的抑制活性,同时对宿主细胞的抑制作用很小。分离组分中的强带电性组分ESPSⅥ活性最高,其TI值达3125,为阳性对照药病毒唑的40余倍。     

超声波降解紫球藻胞外多糖研究

通过测定溶液粘度,判断超声波降解紫球藻(Porphyridium cruentumi)胞外多糖的效果。运用均匀设计对超声波降解紫球藻胞外多糖的影响因素(处理振幅、时间、脉冲)进行优化,获得超声波处理的最佳条件:振幅39%、处理时间245s和脉冲9.5s。在最佳条件下,胞外多糖的粘度为2.98mm²/s,与预测值一致。采用DPS软件对实验结果进行二次多项式分析与拟合,并对模型和回归系数进行显著性检验,建立了以胞外多糖粘度为目标的回归方程式。     

紫球藻胞外多糖的分离纯化与性质分析

采用Q Sepharose Fast Flow凝胶,从紫球藻胞外多糖（ESPS）获得3个组分ESPS0,ESPS1.0和ESPS1.6,它们经过Sephadex G200凝胶过滤纯化后,紫外光谱、红外光谱、氨基酸分析、单糖组成分析表明,ESPS0中未测出蛋白,ESPS1.0和ESPS1.6的蛋白含量分别为7.8%和7.6%,三者硫酸基含量分别为16.3%,11.6%和8.3%,同时分别在201 nT,207 nm和199 nm处出现特征吸收峰,而在280 nm和260 nm波长处无吸收峰.ESPS0、ESPS1.0中单糖的连接方式为β-糖苷键,而ESPS16中主要为α-糖苷键.ESPS1.0、ESPS1.6都含有十七种氨基酸.三种组分都是由D-木糖、D-葡萄糖、D-甘露糖、D-艾杜糖以不同的比例所组成.     

脱落酸对紫球藻（Porphyridiu sp.）生长代谢的影响

为优化培养条件和提高紫球藻(Porphyridiu sp.)生物量及代谢产物产量,研究了7种浓度的脱落酸(ABA)在三种氮素营养水平下对紫球藻生长的影响,结果表明,ABA浓度在0.01～1.0 mg·L-1范围内,外源ABA浓度的升高有利于紫球藻增殖,对紫球藻的干重、叶绿素a含量、蛋白质含量、胞外多糖含量、藻红素含量都有一定的促进作用;但在ABA浓度在10 mg·L-1时,则呈递抑制趋势.其中中氮水平下外源ABA浓度1.0 mg·L-1是紫球藻生长的最佳浓度,最有利于紫球藻生物量及代谢产物的积累,此时紫球藻的生物量为3.463 mg·mL-1,胞外多糖含量最高,占藻体干重的34.31%.在高氦营养水平下还能体现ABA对紫球藻生长所起的作用,而低氮下生长的紫球藻对ABA的作用不敏感.     

Wachstum und Bildung extrazellulärer Polysaccharide in Batch-Kulturen von Porphyridium aerugineum,Rhodophyceae

An batch-Kulturen von P. aerugineum im Algen-Lichtthermostaten und in 5-Liter-Rohren mit Innenbeleuchtung wurde der Einfluß einzelner Nährlösungskomponenten (SO₄-, Mg- und Mn-Gehalt, Tricin- und Tris-Puffer, pH-Wert), sowie von Beleuchtungsstärke und Durchlüftungsintensität auf das Wachstum und auf die Bildung extrazellulärer Polysaccharide untersucht. Beide Prozesse werden durch diese Faktoren verschieden und teils gegensinnig beeinflußt. Als Maß für die Produktion extrazellulärer Polysaccharide diente die Viskosität der Algensuspensionen, für die Wuchsleistung das Trockengewicht. Die für die Bildung extrazellulärer Polysaccharide optimale Sulfatkonzentration der Nährlösung ist doppelt so hoch als die für optimales Wachstum benötigte. Sehr viskose Suspensionen ergeben sich, wenn die molare Mg-Konzentration nur die Hälfte der SO₄-Konzentration des Mediums beträgt. Einseitige Erhöhung der Mg-Konzentration fördert das Wachstum, mindert aber die Bildung extrazellulärer Polysaccharide erheblich. Stark viskositätssenkend bei unverminderter Wuchsleistung wirkt eine Erhöhung des Mn-Gehaltes der Nährlösung. Eine spezifisch viskositätsfördernde Wirkung über die pH-Wirkung hinaus kommt dem Tricingehalt des Mediums zu. Bemerkenswert ist auch das hohe Beleuchtungsoptimum von P. aerugineum. Besonders hohe Wuchsleistungen bei intensiver Produktion extrazellulärer Polysaccharide wurden in den stark belüfteten 5-Liter-Rohren mit Innenbeleuchtung erzielt. Die Ergebnisse werden auch in Hinblick auf eine Möglichkeit zur Gewinnung von gallertigen Wandsubstanzen für industrielle Zwecke diskutiert.     

Swirling flow implementation in a photobioreactor for batch and continuous cultures of Porphyridium cruentum

Light is the main limiting factor in photoautotrophic-intensive production of microorganisms, and improvement of its use is an important concern for photobioreactor design and operation. Swirling flows, which are known to improve mass and photon transfers, were applied to annular light chambers of a photobioreactor and studied by simulation and microalgal culture. Two hydrodynamic conditions were compared: axial flow generating poor radial mixing, and tangential flow generating three-dimensional swirling motion. Batch and continuous cultures of the Rhodophyte Porphyridium cruentum were performed in a 100-L, 1.5-m(2), fully controlled photobioreactor with eight light chambers. The inlet design of these chambers was modified to create the hydrodynamic conditions for comparison. Various intensities of swirling motion were used, characterized by the velocity factor (VF), defined as the ratio between annular chamber flow and inlet aperture sections. Experiments were performed within the range of photon flux densities (PFD) optimizing the yield of light energy transformation into living substance for the species and the temperature used. Culture kinetics with swirling flows generated by apertures of VF = 2, 4, and 9 were compared with pseudoaxial VF = 2 chosen as reference. Batch cultures with VF = 4 swirling flow showed no significant difference, whereas continuous cultures proved more discriminating. Although no significant difference was obtained for VF = 2, a 7% increase of steady-state productivity and a 26% decrease in time required to reach this steady state were obtained with VF = 4 swirling flow. This beneficial effect of swirling flow could have accounted for increased mixing. Conversely, VF = 9 swirling flow resulted in a 9% decrease of steady-state productivity and a 9% increase in the time required to reach this steady state, a negative effect that could have accounted for increased shear stress. CO(2) bioconversion yield at steady state showed a 34% increase for VF = 4. These results suggest that swirling motion makes microalgal cultures more efficient, provided that the resulting adverse effects remain acceptable. Experimental investigation was completed by a theoretical approach in which simulation of continuous cultures of P. cruentum was based on the hydrodynamic conditions achieved in the photobioreactor. Although the results obtained with pseudoaxial flow were correctly predicted, simulations with swirling flow showed a marked enhancement of productivity not observed experimentally. The influence of side effects induced by increased mixing (particularly hydrodynamic shear stress) was considered with respect to modeling assumptions. Comparison of experimental results with theoretical simulation provided a better understanding of the mixing effect, a key factor in improving the efficiency of such bioprocesses.     

Photoperiodic regulation of seasonal responses in Indian weaver bird (Ploceus philippinus)

Photoperiod (=day length) is the vital factor for the regulation of behavioral and physiological activities in many avian species. This study investigated the seasonal cycles of testicular growth and secondary sexual characteristics of Indian weaver bird under natural day length (NDL) and the effects of duration and intensity of light on photoperiodic induction. In the first experiment, groups of birds (n = 7 each) were exposed to under NDL in April 2008 and May 2009 for 8 and 12 months, respectively. In second and third experiment, birds (n = 6 each group) were exposed to different photoperiods (11.5L:12.5D, 12L:12D, 13L:11D, and 15L:9D) at the same (500 lux) light intensity, and to 13L:11D at different light intensities (10-, 50-, 500-, and 800-lux). Observations on testis size, molt, and plumage score were recorded 2-week (molt and plumage) or at 4-week intervals (testes). Both the NDL groups showed similar seasonal cycles of testicular growth-regression and secondary sexual characteristics. Second and third experiments suggest that the photoperiodic induction was depending upon duration and intensity of the light. Birds showed testicular growth-regression cycle followed by molt and plumage color change only under 13L:11D and 15L:9D and only 500- and 800-lux under 13L:11D photoperiod but not under 11.5L:12.5D and 12L:12D and 10- and 50-lux light intensities. Pre- and post-nuptial molting on body feathers were progressed with gonadal stimulation–maturation and regression cycle under 13L:11D and 15L:9D. Results under different light–dark cycles suggest that day length of about 12 h or more and above the threshold level of light intensity are essential for the induction of photoperiodic responses.     

紫球藻及其多糖抗菌性能初探

本文报道紫球藻提取液及其多糖溶液抗病原细菌、真菌性能的研究结果。采用管碟法分别测定紫球藻培养分泌物、藻体破碎后的水提物和醇提物、胞外多糖和胞内多糖水溶液等的抗菌能力。结果表明,各样品均有一定抗菌能力,紫球藻水提物和胞外分泌物抗菌性能较强。经比较分析,多糖是抗菌作用的主要组分。紫球藻提取物及其多糖对革兰氏阳性细菌(特别是对金黄色葡萄球菌和八叠球菌)的抑菌作用较为明显,具有一定开发价值。     

紫外线对紫球藻的生物学效应

采用紫外线辐照紫球藻,处理剂量分别为10s、20s、30s、60s、90s、120s和180s。结果表明,10s剂量对紫球藻有促长作用,20s及30s对藻细胞生长和各项生理生化指标影响不显著,30s以上处理对其生长以及代谢产物的合成与分泌有抑制作用,且照射时间越长,抑制越强烈,紫球藻对紫外线的耐受极限为120s。结果还表明,高剂量(20~90s)照射能增加单细胞代谢产物含量;经紫外线辐照后的藻细胞第二代培养物在生长速度、叶绿素a含量、胞外多糖和β-胡萝卜素产量等与对照组相似。     

光照对蒙古裸腹溞摄食强度的影响

利用细胞计数法对不同光照强度、光谱成分、光周期对蒙古裸腹摄食强度的影响进行了研究。结果表明 :光强在 70 0 0 lx时蒙古裸腹对小球藻的摄食强度最高 ,成的滤水率和摄食率分别为 0 .5 2 8± 0 .0 0 9ml/ (ind· h)和 1.113± 0 .0 15μg C/ (ind· h) ,幼的滤水率分别为 0 .4 95± 0 .0 14 ml/ (ind· h)和 1.0 5 1± 0 .0 0 8μg C/ (ind· h)。在蓝光下蒙古裸腹对小球藻的摄食强度明显高于绿光组。光周期 L∶ D为 12∶ 12时蒙古裸腹对小球藻的摄食强度最高 ,成和幼的滤水率分别为0 .4 6 1和 0 .35 5 ml/ (ind· h) ,摄食率分别为 1.2 4 2和 1.0 0 5μg C/ (ind· h) ,而在 L∶ D为 0 :2 4时摄食强度最差 ,成和幼的滤水率分别为 0 .0 70和 0 .0 5 0 ml/ (ind· h)。在自然光照下 ,蒙古裸腹对小球藻的摄食强度在 7∶ 0 0～ 11:0 0和 15 :0 0～ 19:0 0最高     

硒化紫球藻胞外多糖组成与结构的初步分析

通过在ASW培养基中加入适量的亚硒酸制备硒化紫球藻胞外多糖,经分离纯化、纯度鉴定后,利用下列手段对其进行分析:通过紫外可见光谱扫描、红外光谱扫描了解其结构信息;通过高效液相色谱对其单糖组分进行分析;通过硫酸-咔唑法测定其糖醛酸含量;通过硫酸比浊法测定其硫酸根含量,等。Se-PSP和PSP一样,分离后分别得到两种成分,紫外光谱也和PSP相似,不含有蛋白质和核酸;红外光谱显示Se-PSP中Se可能取代了C-H上的H和SO42-中的S;HPLC显示其单糖组分种类相似,含量稍有差别;另外,PSP和Se-PSP所含的糖醛酸含量没有统计学差异,Se-PSP所含SO42-比PSP少。