佐夫色绿藻高产虾青素突变体的筛选及虾青素合成机理研究

为了提高佐夫色绿藻(Chromochloris zofingiensis)细胞内虾青素含量,研究通过甲基磺酸乙酯诱变构建了含有20000个单克隆的突变体库,并筛选出一株高产虾青素的突变体12C10。在异养正常培养条件下,当葡萄糖耗完时,12C10虾青素含量比野生型提高74%;缺氮诱导第4天时,虾青素含量比野生型高25%。利用广泛靶向代谢组学分析在正常培养条件下12C10与野生型在代谢物水平上的差异。与野生型相比,12C10中除谷氨酸外的氨基酸及脂肪酸的合成普遍下降,但是谷氨酸的水平显著提高。氨基酸和脂肪酸合成减少为虾青素合成提供更多的碳骨架、NADPH和ATP。谷氨酸的积累可能一方面刺激了磷酸戊糖途径中6-磷酸葡萄糖脱氢酶的活性促进NADPH的产生,另一方面导致氧自由基产生促进虾青素合成。代谢物组分析结果还表明12C10中虾青素合成的增强可能与乙烯合成的增强有关。研究为进一步通过代谢调控提高C.zofingiensis虾青素含量奠定了基础,对指导C.zofingiensis虾青素积累新工艺的开发具有重要意义。

不同诱导条件强化佐夫色绿藻积累虾青素

佐夫色绿藻具有多种营养生长方式且可以在诱导条件下合成虾青素,是一种藻源天然虾青素生产的新型替代微藻。本研究系统探讨了以醋酸钠为碳源时,醋酸钠浓度、补料方式、p H以及乙醇对色绿藻积累虾青素的影响,并在室外300L管道光生物反应器中对上述优化的诱导条件进行放大验证。结果表明:采用2.5 g/L醋酸钠、每6 d补加2.5 g/L醋酸钠、培养基p H控制在6.5~8.3和添加2%乙醇时,虾青素的含量最高为3.71 mg/g,较优化前的虾青素的含量提高了31.71%,产量为5.37 mg/L。在室外300 L管道光生物反应器中,其最高虾青素含量和产量分别为3.44 mg/g和1.10 mg/L。本研究采用不同诱导条件强化佐夫色绿藻胞内虾青素的积累,验证了佐夫色绿藻在户外光生物反应器规模诱导培养以生产虾青素的可行性。

佐夫色绿藻高产虾青素的诱导条件及发酵工艺优化

本研究了不同诱导条件及光发酵罐培养工艺对混养佐夫色绿藻的影响,通过参数优化提高了藻细胞生物量和虾青素积累量。本研究首先系统地比较了在摇瓶系统中不同混合碳源和过氧化氢浓度对佐夫色绿藻的生长和虾青素积累的影响,并在光发酵罐中研究了恒定高光强、低光强-高光强以及低光强-高光强-补加过氧化氢三种不同发酵工艺对佐夫色绿藻积累虾青素的影响。结果表明:采用20 g/L葡萄糖和2.50 g/L醋酸钠作为混合碳源取代单一碳源,可以获得最高6.50 g/L生物量,并且添加107.50 mg/L过氧化氢可以将虾青素含量提高到3.23 mg/g,产量最高为72.47 mg/L,是空白组虾青素产量的1.80倍,有效促进了佐夫色绿藻细胞生长和虾青素积累。在5 L光发酵罐中,以20 g/L葡萄糖和2.50 g/L醋酸钠作为混合碳源培养佐夫色绿藻,通过低光强-高光强-补加过氧化氢的组合方式,可获得较优的虾青素含量(3.82 mg/g)和产量(41.41 mg/L),相较于恒定高光强培养,分别提高了36.92%和92.96%。本研究通过诱导条件和发酵工艺优化有效提高了混养佐夫色绿藻生物量和虾青素产量,为利用光发酵罐培养色绿藻生产虾青素提供基础。

营养强化混养条件下提高色绿藻生物量和虾青素产量

本研究在混养条件下,系统地比较了葡萄糖浓度、氮源种类以及不同碳氮比对色绿藻生物量和虾青素产率的作用规律。目的是在短时间内达到最高生物量同时获得较高含量的虾青素,为建立色绿藻高密度快速扩种和诱导积累虾青素应用技术提供科学依据。研究结果表明:在混养条件下,当葡萄糖浓度一定时,硝酸钠是细胞生长所需的最优氮源,6 d可达到最高生物量浓度9.23 g/L,平均比生长速率为0.24/d,虾青素产量为12.38 mg/L,虾青素占总类胡萝卜素的比例高达46.94%。至于不同碳氮比、葡萄糖浓度对色绿藻生物量和虾青素生产的影响,当葡萄糖浓度为30 g/L、C/N比为34为细胞生长的最优条件,生物量浓度最高为11.28 g/L,平均比生长速率高达0.32/d;虾青素含量显著优于其他组(p<0.05),虾青素的产量为21.77 mg/L,虾青素占总类胡萝卜素的比例进一步提高到52.71%。本研究结果对于色绿藻高密度快速生长并积累大量虾青素的放大技术开发具有重要的指导意义。

Growth performance,digestive enzyme activities and serum nonspecific immunity of the red tilapia(Oreochromis mossambicus×Oreochromis niloticus)feddiets supplemented with ultrafine powder of Enteromopha prolifera

The present study evaluated ef fects of ultrafine powder of the green macroalgae E nteromopha prolifera as dietary supplement on growth performance, digestive enzyme activities and serum nonspecific immune responses of the red tilapia(O reochromis mossambicus × Oreochromis niloticus). The red tilapia were fed five diets supplemented with different levels of E. prolifera ultrafine powder as well as a control diet containing no E. prolifera for seven weeks(Diets 0–6 contained 0(control), 10, 20, 30, 40 and 50 g/kg of E. prolifera ultrafine powder, respectively). The results showed that diets supplemented with E. prolifera ultrafine powder generally improved growth, immunity and digestive enzyme activities of the red tilapia. In particular, the fish fed the diet incorporated 50 g/kg (5%) E. prolifera ultrafine powder(Diet 5) achieved the highest percentage weight gain, specific growth rate and the condition factor(increased by 15.4%, 8.0% and 5.7%, respectively when compared to the control). Feeding the diet also led to significantly increases( P <0.05) in serum nonspecific immune responses, including total superoxide dismutase, acid phosphatase, alkaline phosphatase, lysozyme activities and serum total protein(increased by 19.4%, 48.1%, 29.5%, 30.3% and 8.7%, respectively) as well as digestive enzyme activities of erepsin, gastric amylase, gastric lipase, pepsin, intestinal amylase and gastric lipase(increased by 15.7%, 33.3%, 16.3%, 21.3%, 52.3% and 28.2%, respectively) than those of the control. Based on these results, it is recommended that the inclusion level of E. prolifera ultrafine powder in the diet of the red tilapia should be 50 g/kg(or 5%).

Combined effects of light intensity and NH_4^+-enrichment on growth, pigmentation, and photosynthetic performance of Ulva prolifera(Chlorophyta)

The aim of this study was to investigate the effects of light intensity and enhanced nitrogen supply on the growth and photosynthesis of the green-tide macroalga, Ulvaprolifera. Thalli of U. prolifera were grown in natural or NH^-enriched seawater under two different light intensities for 7 days, and then the growth rate, pigmentation, and photosynthetic performance of the thalli were evaluated. The results show that the relative growth rate （RGR） was markedly higher under the high light level than under the low light level. Enrichment with NH~ enhanced the RGR under high light intensity, but did not affect RGR under low light intensity. In low light conditions, NH;-enrichment resulted in a marked decrease in the maximal photosynthetic rate （Pro） and the maximum carbon fixation rate （Vmax）, but it did not affect the half saturation constant for carbon （K0.5） or the ratio of Vmax to K0.5, which reflects the carbon acquisition efficiency. In high light conditions, Pm, K05, and the dark respiration rate （Rd） increased under NHI enrichment, but Vmax and the Vmax/Ko5 ratio decreased. Regardless of the light intensity, NH^＋4-enrichment did not affect the apparent photosynthetic efficiency （a）, which reflects the ability of the alga to use light energy at low light levels. Under both low and high light intensities, the chlorophyll a （Chl a）, chlorophyll b （Chl b）, and carotenoids （Car） contents in thalli were higher in NH1-enriched than in natural seawater, except that there was a decrease in the Chl b content of thalli in NH1-enriched seawater under low light intensity. Therefore, NH^＋4 enrichment improved the growth and photosynthetic performance of U. prolifera under high light intensity, but not under low light intensity. We discuss the possible mechanisms underlying these physiological responses.

Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera （Ulva prolifera O. F. Miiller）, a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10072 high-quality expressed sequence tags （ESTs）. These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum （Ulvophyceae）.

Marine Green Algae as a Supplement for Chlorophyll and Other Nutrients in Vigna Radiata under UV-C Radiation-Induced Stress

The potential protective effect of marine green algae （Codium iyengerii） was examined in UV-C treated seedlings of Vigna radiata. The study comprises of three treatments of UV-C radiation （100-290 nm） dose for one min in alternative days. This results in deformed morphological parameters, including： decrease in plant height, fresh mass of leaves, shoots and roots, as well as leaf areas, which may be attributed with decreased in the relative growth rate, carbohydrate, amino acids, and protein contents of plant. A drastic effect of UV-C radiation was found on the photosynthetic apparatus where increase in red pigmentations on the leaves surface indicates the presence of UV-C absorbing pigments instead of chloroplast pigments. Visible spectrum of leaves chlorophyll showed reduced concentration of visible absorbing pigments which showed the deleterious effect of these radiations on physiological processes of seedlings. These negative effects of UV-C radiation on plant growth were found to be decreased by the application of green seaweed （Codium iyengerii）, and absorption spectrums of chloroplast contents showed that UV-C radiation inducing damages were appropriately managed by enhanced concentration of seaweeds which significantly increased morphological and physiological parameters like leaf, stem, root biomass, and plant height under UV-C radiation were observed.