氮、锰、硫缺乏对蛋白核小球藻Chlorella pyrenoidosa光合产氢及其生长的影响

本实验通过研究缺氮、缺锰和缺硫对蛋白核小球藻Chlorella pyrenoidosa产氢的影响,发现缺氮、缺锰及缺硫条件下该藻均能产氢,但在缺氮条件下产氢量最高,约为88.613μL H2/mgChla,分别是对照组、缺锰和缺硫实验组产氢量的4.61倍、1.92倍和3.63倍。通过对光合、呼吸及生长的比较研究,发现缺锰对该藻光合、呼吸及生长的影响要小于缺氮和缺硫;与正常培养条件相比,缺锰、缺硫抑制藻细胞的光合放氧和生长,对呼吸影响小,而缺氮不仅最大程度抑制光合放氧和生长,同时使呼吸作用增强,这为进一步优化该藻产氢条件及研究其产氢机制提供了线索。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoidosa No.2。研究了培养基组成及培养条件对其细胞生长和油脂积累的影响。结果表明, 最适培养基组成为(g/L)：葡萄糖 20, 甘氨酸 0.08, MgSO4·7H2O 0.4, K2HPO4 1.0, FeSO4·7H2O 0.004; 适宜的培养温度、初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和 650 Lux。在上述优化条件下培养7 d, Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L 和 40.15%提高到6.56 g/L和59.90%, 油脂产量提高了162%。Chlorella pyrenoidosa No.2能以木糖为碳源产油脂, 可望用于以木质纤维素等可再生生物质资源为原料生产油脂。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 不饱和脂肪酸含量达71％左右, 可作为生产生物柴油的原料。     

增强UV-B对蛋白核小球藻中NO释放的影响

研究了蛋白核小球藻在增强UV—B辐射下NO信号的产生。结果表明：NO释放量与UV—B的强度相关。在藻类细胞培养液中加入NOS的竞争性抑制剂硝基精氨酸(LNNA),不能抑制NO的释放。加入NO清除剂乙酰半胱氨酸(NAC),能明显减少NO的释放。在无氮和有氮培养基中NO同样的释放。这些结果说明NO生成途径不经过依赖L-精氨酸的NOS和依赖NO3^-和硝酸还原酶,NO的底物不是L-精氨酸和NO3^-在无氮和有氮培养基中同样有NO3^-的形成,NAC减少NO2^-的释放,说明生成了NO2^-有可能是NO释放后生成的。     

锌胁迫对小球藻抗氧化酶和类金属硫蛋白的影响

通过对抗氧化酶活性和类金属硫蛋白的测定,考察在0、5、10、20、50和100 μmol/L Zn²⁺(氯化锌)胁迫下锌对普通海洋小球藻的生物学影响。结果表明:不同浓度Zn²⁺均能抑制小球藻的生长,当Zn²⁺浓度大于10 μmol/L时,小球藻生物量随培养时间延长而迅速下降;过氧化物歧化酶 (SOD)活性随Zn²⁺胁迫浓度的增加而增加,当Zn²⁺浓度为50 μmol/L时SOD活性达到最大,但继续增加Zn²⁺胁迫浓度反而导致SOD活性下降;而过氧化物酶 (POD)活性则随着Zn²⁺胁迫浓度的增加而降低。同时,实验发现藻细胞内有两种主要的锌结合形态,其中Zn结合类金属硫蛋白(Zn-MT-like)与兔肝金属硫蛋白(MT)的分子量相近,且随着Zn²⁺胁迫浓度的增加而出现规律性地增多。因此,藻细胞内Zn-MT-like蛋白的诱导量可作为小球藻受Zn²⁺胁迫的响应指标。     

不同氮源对蛋白核小球藻生长、色素和中性脂肪积累的影响

单细胞微藻在生长发育过程中,所积累的中性脂肪具有潜在的生物燃料价值。不同氮源对藻类的生长具有显著影响。研究了氨态氮、尿素氮和硝酸态氮对蛋白核小球藻生长、色素和中性脂肪积累的影响。结果显示,不同氮源对培养液pH有显著影响,以氨态氮为氮源导致培养液pH降低,而硝酸态氮导致培养液pH升高,培养液pH的波动可被添加的Hepes所稳定,并促进以氨态氮为氮源的蛋白核小球藻的生长。尿素氮对蛋白核小球藻生长、色素积累的效果优于氨态氮和硝酸态氮,硝酸态氮在中性脂肪积累上优于尿素氮和氨态氮,添加Hepes对氮饥饿后蛋白核小球藻的中性脂肪积累无显著影响。     

蛋白核小球藻凝集素的分离纯化及部分性质研究

蛋白核小球藻藻粉的PBS抽提液经硫酸铵二步分级沉淀 ,再经DEAE Sepharose和SephadexG 10 0层析 ,从中分离纯化得到蛋白核小球藻凝集素 (CPL)。经测定 ,该凝集素为单个亚基的蛋白质 ,相对亚基分子量为 1 4× 10 4 — 1 5× 10 4 ,分子中不含糖。在氨基酸组成中 ,苯丙氨酸 (Phe)的含量最高 ,其次是天冬氨酸 (Asp)和谷氨酸 (Glu) ,不含组氨酸 (His)。CPL能够凝集兔、绵羊及鸽子红细胞 ,其中对兔红细胞的凝集活性最大 ,最低浓度为 6 88μg/mL ,对鸡、鸭及人红细胞 (A型、O型及B型 )无凝集活性。卵黏蛋白和 7种单糖对CPL的凝血活性具有抑制作用。CPL具有很好的热稳定性 ,在 90℃处理 10min不失活。     

绞股蓝提取液对蛋白核小球藻的化感效应及影响机理研究

以中草药植物绞股蓝[Gynostemma pentaphyllum(Thunb.) Makino]为化感供体材料,研究其不同浓度的提取液(0、5、10、25、50 g/L)对蛋白核小球藻(Chlorella pyrenoidesa)生长及生理生化特征的化感效应。结果表明:(1)绞股蓝提取液对蛋白核小球藻生长均具有抑制作用,其抑制作用随提取液质量浓度增大和培养时间延长均呈增强趋势,且25 g/L绞股蓝提取液培养15 d时的抑制率达到最大(79.41%)。(2)各浓度绞股蓝处理组蛋白核小球藻细胞内的叶绿素a含量均低于对照组,且随着提取液浓度升高以及处理时间延长叶绿素a含量较对照的降低量越多,表明蛋白核小球藻光合作用受到的影响也越大。(3)绞股蓝处理组蛋白核小球藻细胞的膜透性(吸光度OD_(264))显著高于对照,且膜透性随着提取液浓度增大而增强;高浓度提取液处理下,藻细胞内部的可溶性蛋白质(OD_(280))及核酸(OD_(260))含量均显著高于对照,且随着处理时间延长,细胞膜透性增大,细胞内部的可溶性蛋白质及核酸向胞外渗透增多。研究发现,绞股蓝提取液能够抑制蛋白核小球藻生长,并随着提取液质量浓度增大而增强;绞股蓝提取液能促进藻细胞叶绿素分解、增加细胞膜透性,引起可溶性蛋白质和核酸向胞外渗透量升高,导致藻细胞结构受损,代谢功能紊乱,从而达到化感抑制作用。     

柳树叶浸提液对蛋白核小球藻的化感作用

通过对蛋白核小球藻进行细胞密度的测定,分析了不同浓度的柳树(Salix babylonica)叶浸提液对蛋白核小球藻(Chlorella pyrenoidosa)生长和细胞形态的影响。结果表明：柳树叶浸提液对蛋白核小球藻具有化感作用,不同浓度的浸提液化感作用强度不同;作用96 h的半效应浓度(EC₅₀,_{96 h}值)为11.82 g·L^-1,较低浓度(10和20 g·L^-1)浸提液对蛋白核小球藻表现出先抑制后促进的作用特性,而较高浓度（30 g·L^-1）浸提液则始终表现出抑制效果;10、20和30 g·L^-1浸提液对蛋白核小球藻的最大抑制率分别为37.5%、39.3%和56.5%;柳树叶浸提液作用下,部分蛋白核小球藻细胞平均颗粒体积增大,颜色变透明,膨胀后溶解;低浓度浸提液处理组少数细胞聚集在一起,高浓度浸提液处理组细胞大量聚集,形成多细胞群体,多数沉于瓶底,沉降性能增强。     

蛋白核小球藻发酵产油脂的研究

从5种不同来源的小球藻中筛选到1株油脂产量较高的蛋白核小球藻Chlorella pyrenoi-dosa No.2.研究了培养基组成及培养条件对其细胞生长和油脂积累的影响.结果表明,最适培养基组成为(g/L):葡萄糖20,甘氨酸0.08,MgSO4·7H2O 0.4,K2HPO4 1.0,FeSO4·7H2O 0.004;适宜的培养温度,初始pH、摇床转速和光照强度分别为28℃、6.0、130 r/min和650 Lux.在上述优化条件下培养7 d,Chlorella pyrenoidosa No.2的生物量和油脂含量分别由优化前的3.73 g/L和40.15%提高到6.56 g/L和59.90%,油脂产量提高了162%.Chlorella pyrenoidosa No.2能以木糖为碳源产油脂,可望用于以木质纤维素等可再生生物质资源为原料生产油脂.气相色谱分析表明该油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达71%左右,可作为生产生物柴油的原料.     

定向驯化对异养蛋白核小球藻硒的耐受性及富集能力的影响

硒(Se)是生物体必不可少的微量元素,硒缺乏会导致人产生克山病、大骨节病等疾病,缺硒也会给畜牧业带来巨大损失。目前的补硒产品存在硒含量和生物利用度低、安全性差等问题,而通过小球藻培养可获得生物利用度高、安全性好的有机硒,因此是非常有应用前景的补硒产品。首先,为了获得对硒的耐受性和富集能力更强的藻种,研究通过定向驯化的方式逐步提高培养基中Na₂SeO₃浓度来驯化蛋白核小球藻,并对驯化时间和驯化过程中Na₂SeO₃的浓度梯度进行了优化。结果表明,驯化后的藻种对硒的耐受性和富集能力明显提高。在5L发酵罐中,驯化后的藻株可以耐受40mg/L的Na₂SeO₃,胞内有机硒合成速率提高了175.6%。之后,在5L发酵罐中进一步优化了硒的补加方式,在异养培养过程中分批补料添加40mg/LNa₂SeO₃时,最终获得的蛋白核小球藻细胞干重达106.4g/L,有机硒含量为1227mg/kg,有机硒合成速率为1.36mg/(L·h)。研究结果与已有蛋白核小球藻异养富硒文献报道的最高细胞密度75g/L和最高有机硒含量560mg/kg相比分别提高了41.9%和119.1%。上述结果表明,通过定向驯化的方法,可大大提高蛋白核小球藻对硒的耐受性和富集能力。     

Extracellular Carbohydrates and Polysaccharides of the Alga Chlorella pyrenoidosa Chick S-39

In the growing culture of the thermophilic alga Chlorella pyrenoidosa Chick S-39, the amount of extracellular carbohydrates in the medium reached 5–17% of their content in the cells and 20–40% of the total content of extracellular organic matter. Experiments with the enrichment and synchronous algal cultures showed that the accumulation of extracellular carbohydrates and polysaccharides in the media occurred due to their release from the cells, rather than to cell lysis, and depended on cell photosynthetic activity and reproduction. Chromatographic determination of free sugars revealed the presence of saccharose, glucose, and fructose in the culture medium. Extracellular carbohydrates in C. pyrenoidosa cultures were represented mainly by water-soluble polysaccharides containing galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.     

Regulation of the Size of Light-Harvesting Antennae in Response to Light Intensity in the Green Alga Chlorella pyrenoidosa

Changes in size of the light-harvesting Chl-protein complex(LHC) induced by changes in light intensity were studied withthe green alga Chlorella pyrenoidosa. The Chi a/b ratio, whichis correlated with the size of LHC, varied over a wide range(2.5–5.0) when the light intensity for autotrophic growthwas changed. Comparison of properties of LHC II isolated fromcells grown under light of high and low intensity indicatedthat the large difference in Ch1 a/b ratios in cells grown underlight of different intensities is due mainly to changes in levelsof LHC. Reduction in levels of LHC under light of high intensitydid not occur when proliferation of cells was suppressed. Thisresult indicates that reduction in levels of LHC is not attributableto acceleration of the degradation of LHC under light of highintensity. Stimulation of formation of LHC occurred even underlight of high intensity when formation of photosystems was suppressedby chloram-phenicol (CAP). Analysis of the CAP-induced formationof LHC indicated that (1) such formation of LHC was regulatedby light intensity, being less active under higher intensity,and (2) the suppressive effect of gabaculine, an inhibitor ofthe synthesis of porphyrin, and thus of Ch1, was greater underlight of high intensity while the suppressive effect of cycloheximide,an inhibitor of the synthesis of apoprotein, was slightly greaterunder light of low intensity. The results described in thisreport indicate that (1) intensity-induced changes in the sizeof LHC in Chlorella pyrenoidosa are due to regulation of theassembly of LHC and (2) the regulation occurs primarily at thelevel of the synthesis of Ch1. (Received June 1, 1989; Accepted August 21, 1989)     

Effects of Environmental pH on the Internal pH of Chlorella pyrenoidosa, Scenedesmus quadricauda, and Euglena mutabilis

The effect of external pH on two laboratory-cultured acid-intolerant species (Chlorella pyrenoidosa Chick and Scenedesmus quadricauda Turp. Bréb.) and one acid-tolerant species from a natural population (Euglena mutabilis Schmitz) was examined by measuring internal pH. These measurements were made with the weak acid ¹⁴C-dimethyloxazolidine-2,4-dione after cells had been incubated for 2 and 6 hours at external pH levels from 3.0 to 8.0. Photosynthetic and respiration rates of the three species were also measured over the range of external pH levels.     

Effects of Phenylethyl Alcohol on Chlorella pyrenoidosa

Asynchronous as well as synchronous cultures were used to study the effect of phenylethyl alcohol on Chlorella pyrenoidosa. The following observations were made:

• 1 Autotrophic growth and the synthesis of DNA and chlorophyll were inhibited more than 80% by 8.4 mM phenylethyl alcohol when added to synchronous suspensions containing autospores.
• 2 Autospore formation did not occur when fully grown cells capable of division were transferred to 4.2 mM phenylethyl alcohol.
• 3 Photosynthetic oxygen evolution of asynchronous cultures was inhibited more than 70 % by 21 mM phenylethyl alcohol.
• 4 Endogenous respiration in the dark was stimulated by 8.4 mM, while higher concentrations were inhibitory.
• 5 Glucose respiration in the dark was inhibited by all concentrations in the range from 8.4 mM to 21 mM. Assimilation of glucose in darkness was retarded by phenylethyl alcohol at concentrations which gave maximal stimulation of endogenous respiration.

It is concluded that phenylethyl alcohol is not a specific inhibitor of DNA synthesis. The prime effect appears to be on respiration and photosynthesis.     

Heterotrophic Growth and Production of Xanthophylls by Chlorella pyrenoidosa

The growth and level of xanthophylls of several representative species of green algae were investigated as a possible source of pigmentation for the egg yolk and broiler markets. Chlorella pyrenoidosa 7-11-05 was selected for fermentation studies because of its high level of xanthophylls and wide temperature range for growth. The heterotrophic metabolism was preferred because of the ease of adaptability to present fermentation equipment. When used as the sole carbon source, glucose was the only sugar, among many tested, that gave appreciable growth in illuminated shaken flasks. A dry cell weight of 90 g per liter and total xanthophylls of 450 mg per liter were obtained from 190 g per liter of glucose monohydrate in 168-hr illuminated shaken flasks. Higher levels of glucose decreased yields. In combination with glucose, monosaccharides, such as fructose and galactose, were readily assimilated. The 7-11-05 strain was adapted to galactose as the sole carbon source after six vegetative passages. Light of the proper intensity and duration stimulated total xanthophylls approximately 35%. The effect on dry cell weight and total xanthophylls of seven antibiotics added at various levels in shaken flasks was studied. Erythromycin was essentially stable throughout the fermentation and nontoxic up to 25 μg/ml, with only slight toxicity at higher levels. Both erythromycin and ristocetin were effective in controlling a high incidence of bacterial contamination in 30-liter fermentors. With the higher agitation and aeration rates possible in 30-liter fermentors, dry cell weights in excess of 100 g per liter and total xanthophylls of 467 to 512 mg per liter were readily obtained from 230 to 260 g per liter of glucose in 162-hr illuminated batch-type fermentations. Continuous-feed runs yielded a dry cell weight of 302 g per liter and total xanthophylls of 650 mg per liter from 520 g per liter of glucose. The type of Chlorella cell produced was an important consideration with respect to the availability of the xanthophylls in pigmenting egg yolks and broilers.     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

光照强度、温度、pH、盐度对小球藻（Chlorella）光合作用的影响

利用测定净光合放氧速率的方法研究了光照强度、温度、pH、盐度对小球藻（Chlorella sp.XQ-200419）和海洋小球藻（Chlorella marina NJ-016）光合作用的影响。小球藻（Chlorella sp.XQ-200419）的适宜光照强度范围为100～〉1600μmo·lm-2·s-1,光饱和点在500μmo·lm-2·s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为6.5～9.0,最适pH值为7.0;对盐度的适应范围较广,在0～0.6mol/L范围内,随着盐度的升高,净光合放氧速率有下降趋势。海洋小球藻（Chlorella marina NJ-016）的适宜光照强度范围为400～〉1600μmol·m-2·s-1,光饱和点在1400μmo·lm-2.s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为5.0～9.0,最适pH值为8.0;对盐度有很好的适应性,在0～0.6mol/L范围内,随着盐度升高,净光合放氧速率明显上升。小球藻和海洋小球藻的净光合放氧速率随光照强度、温度、pH值和盐度变化的规律,表明了两种小球藻的基本生理生态学特性：能适应较强的光照强度、较高的温度、中性偏碱的环境和较高的盐度。研究结果有助于小球藻培养条件的优化。两种小球藻对光照强度、温度、pH值和盐度变化的反应也有所不同：与小球藻（Chlorella sp.XQ-200419）相比,海洋小球藻（Chlorella marina NJ-016）对光照强度有更好的适应性,对pH值变化有更宽的适应范围,适当提高盐度对其光合作用有明显的促进作用。这表明海洋小球藻（Chlorella marina NJ-016）在快速生长繁殖方面具有更大的潜力,这一研究结果为筛选适合于大量培养的优良藻种提供了依据。