Photon requirement for growth of the diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae)

Abstract Photon requirements for growth (φ_g^?1) of the diatom Phaeodactylum tricornutum were determined under nutrient-sufficient conditions at two photon flux densities corresponding to light limited and near-saturating conditions for growth. The value of φ_g^?1 based on assimilated carbon was light-dependent and varied from 8.8 to 14.0 mol photon mol C^?1 with the minimum value at the lowest photon flux density. These results are lower than might be predicted for microalgal growth based on the Z scheme of photosynthesis. Conversion of these values for carbon fixation to estimates based on oxygen evolution is problematical due to uncertainty over the appropriate assimilatory quotient (Q_a= mol O₂ mol C^?1). Minimum values based on oxygen evolution rates ranged from 6.2 to 7.6 mol photon mol O₂^?1 using a Q_a of 1.41 mol O₂ mol C^?1 obtained by Myers (1980). These estimates are similar to our previous measurements for photosynthesis and indicate a high efficiency for light energy transforming reactions during growth. The values of (φ_g^?1 obtained in this work indicate a number of inadequacies in our understanding of the energetics of microalgal growth and are inconsistent with our present knowledge of photosynthetic energy coupling in plant cells.     

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L^-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G₂ + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G₂ + M phase can accumulate to over 95%. Even after a 48-h treatment with nocodazole (2.5 mg L^-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible. This revised version was published online in August 2006 with corrections to the Cover Date.     

三角褐指藻在磷营养限制胁迫下的补偿生长效应

以三角褐指藻(Phaeodactylum tricornutum)为研究材料,设置了5个磷营养限制处理:磷营养分量分别设为f/2培养基的1/20(P₁)、1/10(P₂)、1/8(P₃)、1/4(P₄)、1/2(P₅),以f/2为对照(P_ck),在磷限制胁迫下培养10d,然后均以相同密度(2.5×10⁵cells·mL^-1)接种在f/2条件下恢复培养16d,测定了三角褐指藻在磷限制胁迫下和恢复培养阶段的生长状况。结果表明,三角褐指藻在受到磷限制胁迫后,细胞密度、蛋白质和可溶性糖含量都显著低于对照(p<0.05);恢复阶段,P₁、P₂和P₃处理组的细胞密度、平均相对生长率及生物量在恢复培养的中前期显著高于对照(p<0.05),最高平均相对生长率分别为0.73、0.70、0.68d^-1,显著高于对照(0.55d^-1)(p<0.05);P₄处理组的细胞密度和生物量与对照无显著差异;P₅处理组的细胞密度和生物量在恢复培养的中前期显著低于对照(p<0.05);随着培养时间的推移,各处理组与对照之间的差异逐渐缩小,处理组和对照的细胞密度、生物量、蛋白质和可溶性糖含量等均无显著差异。     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.     

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors

Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m^?2 d^?1) and EPA (about 0.35 g m^?2 d^?1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m^?2 d^?1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

三角褐指藻对黑暗胁迫的生理响应

为了研究黑暗条件对三角褐指藻生长及生化组成的影响,并探讨三角褐指藻对黑暗环境的生长适应能力,我们对该藻进行12 d的黑暗处理,着重测定了藻细胞密度、生物量、叶绿素a、可溶性糖和蛋白含量等指标。结果表明,三角褐指藻对黑暗环境表现出一定的适应性忍耐能力,在12 d的长期黑暗胁迫条件下依然可以存活,而藻细胞生化组成对黑暗的响应程度很可能是该藻得以维持细胞低水平生长的主要原因。随着黑暗处理时间的延长,三角褐指藻生长状况受抑制的程度增大,其生长及生化组成与对照组相比发生了极显著的变化。实验结束时,黑暗处理下的藻细胞密度和生物量分别降低到2.93×10⁵ cells·ml^-1和0.011 g·ml^-1,仅为对照的8.0%和37.3%。同样地,黑暗环境也明显地抑制了三角褐指藻体内生化物质的合成与积累,黑暗处理12 d时藻细胞的叶绿素a、可溶性糖和蛋白含量分别比对照降低了约89%、87%和85%。研究结果可为海洋微藻种质的筛选、种质资源库的构建及微藻生物资源的综合开发利用提供参考依据。     

Research note: High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis

A high efficiency transformation system was established for the pennate diatom Phaeodactylum tricornutum Bohlin using a plasmid containing fucoxanthin chlorophyll a/c binding protein ( fcp ) promoter/terminator and nitrate reductase ( NR ) promoter/terminator that are derived from the pennate diatom Cylindrotheca fusiformis . The plasmid that contains the zeocin resistance gene ( ble ) with the fcp promoter and enhanced green fluorescent protein gene ( egfp ) with the NR promoter was introduced into P. tricornutum using microparticle bombardment. Transformants (650 ± 58 per 10⁸ cells) were obtained. The yield of transformants was between 1.5 and 130 times higher than previously reported P. tricornutum transformation systems. Four to seven copies of the ble gene were integrated into genomic DNA of the transformants. This high efficiency transformation system of P. tricornutum is expected to provide a powerful tool for high-throughput analysis of gene function using homologous recombination or RNAi.     

Qualitative and quantitative composition of pigments in Phaeodactylum tricornutum (Bacillariophyceae) stressed by iron

The effect of Fe(III) deficiency on qualitative and quantitative changes in pigment composition in Phaeodactylum tricornutum Bohlin was demonstrated by HPLC and AAS. Maximum content of pigments showed the diatom cells incubated at the optimum iron concentration, i.e., 10 M. The contents of chlorophyll a, chlorophyll c ₁+c ₂, fucoxanthin, diadinoxanthin and ,-carotene were 109.99, 20.16, 40.39, 1.29 and 1.48 fg per cell, respectively. The results obtained showed that Fe(III) affected qualitative and quantitative pigment composition in P. tricornutum. The content of individual pigments, proportions between accompanying pigments and their ratios to chlorophyll a were important indicators of phytoplankton response to iron stress. The strong reduction in ,-carotene content, several times (2–5) increase in diadinoxanthin level as compared to ,-carotene, and high amount of diadinoxanthin in relation to chlorophyll a were observed in algae growing at very low Fe(III) concentrations, 0.001 and 0.01 M. The data suggested that phytoplankton pigments could be a potential physiological marker.     

Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.

In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Mixotrophic productivity of the marine diatom Phaeodactylum tricornutum cultured with soluble fractions of rye,wheat and potato

The marine microalga Phaeodactylum tricornutum was cultured semi-continuously with the soluble fractions of wheat, rye and boiled potato flours. Fifteen percent of the culture volume was renewed every 3 d. The cell productivities were 0.9×109 cells/l/d, 1.1×109 cells/l/d and 2.6×109 cells/l/d for wheat, rye and potato respectively. The productivity of the autotrophic control was 1.0×109 cell/l/d. When a soluble fraction of raw potato was added, the productivity was enhanced to 4.1×109 cells/l/d, 2.4 times higher than the autotrophic culture. The high productivity of P. tricornutum with the soluble fractions of Solanum tuberosum suggests its usefulness as a source of nutrients for the production of microalgal biomass.     

UV-B辐射和蒽对三角褐指藻DNA伤害的相互作用

运用生态毒理学和生物化学的方法研究了紫外线和多环芳烃－蒽对三角褐指藻DNA的伤害作用,结果表明,蒽对三角褐指藻的生长有抑制作用;随着蒽浓度的增加,三角揭褐藻DNA损伤程度增加;在蒽浓度固定不变时,随着处理时间的延长,DNA的损伤程度同样提高;在蒽的处理过程中同时伴有紫外线的辐射处理,DNA的损伤程度加剧;蒽处理解除一段时间后,DNA损伤程度未明显减轻、而UV－B处理解除后,DNA的损伤可明显恢复,说明DNA的损伤可在一定程度上指示海洋微藻受蒽伤的程度。     

Growth and photosynthetic rates of Phaeodactylum tricornutum Bohlin in a cyclical light field

Growth rates, cell chlorophyll a concentrations, and carbon uptake rates of Phaeodactylum tricornutum Bohlin grown in cyclical and constant light regimes at three mean irradiances (PFD) were compared. Light cycles with periods 5–38 min were used. Under high PFD the cyclical light regime resulted in both increased and decreased growth rates dependent on the signal frequency. Growth rates were universally depressed under mid and low PFD. Minimal influence of the light field was apparent on cell chlorophyll concentrations. The most marked effect of the fluctuating light was recorded on the carbon uptake rates with maximal influence demonstrated in those samples both grown and incubated in fluctuating light at high as opposed to mid and low PFDs. From the combination of light fields employed, the existence of both short and long term physiological controls of the carbon uptake rates were deduced, these being related to the time scales of carbon uptake and cell growth. A partial explanation to account for the observed increases in carbon uptake in terms of short period non-linear uptake kinetics was derived from two further experiments.     

Direct measurements of nanomolar nitrate uptake by the marine diatom Phaeodactylum tricornutum (Bohlin). Implications for studies of oligotrophic ecosystems

A new automated procedure for nanomolar nitrate analysis was applied to the study of uptake of low nitrate concentrations (< 100 ngat l^–1) by phytoplankton. The precision of this analytical method (± 3 ngat l^–1) made it possible to monitor the absorption of very low quantities of nitrate over short term periods by a low cell-density culture of the marine diatom Phaeodactylum tricornutum, where the levels of particulate nitrogen and chlorophyll were equivalent to those found in oligotrophic areas (0.5 µgat N l^–1 and 0.4 µg l^–1 respectively). By continuous monitoring of nitrate disappearance from the culture medium, we are able to describe accurately the transient uptake responses of the diatom after a spike addition of trace quantities of nitrate and thus to provide new information on the still largely unknown small-scale phenomenon of pulsed nitrate supply in the upper layer of stratified oceans and rapid uptake of these nitrate patches by phytoplankton.The results show that a N-limited culture of Phaeodactylum tricornutum is immediately capable of taking up trace quantities of nitrate (< 100 ngat l^–1) at high rates (0.10–0.14 h^–1) . These initial rates are one order of magnitude higher than the theoretical rates calculated from the Michaelis-Menten equation and are close to the level of V _max (0.15 h^–1) obtained when cells are exposed to saturating nitrate concentrations. This rapid initial uptake would be a considerable advantage in oligotrophic areas where nanomolar nitrate supply is thought to be episodic. The present results suggest that phytoplankton evolve adaptations to utilize the available nitrate at the spatial and temporal scales at which it occurs. On the other hand, we can consider this physiological adaptation as evidence of nitrate pulses in the field which would invalidate the steady-state approach to the oligotrophic ecosystems.     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum (Bacillariophyta)

Responses of the photosynthetic activity of Phaeodactylum tricornutum (Bacillariophyta) to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem (PS) Ⅰ and PS Ⅱ and the values of F6851F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

对硫磷对三角褐指藻核酸和蛋白质合成动态的影响

应用同位素标记法研究了对磷磷对三角褐指灌核酸和蛋白质合成动态的影响。结果表明,低浓度的对硫酸（≤１．５ｍｇ／Ｌ）对三角褐指的生长有刺激作用,而高浓度的对硫磷（≥２．０ｍｇ／Ｌ）却严重抑制三角褐指藻的生长,低浓度划硫磷在促进生长的过程中,藻细胞中蛋白质、ＤＮＡ、ＲＮＡ３种大分子物质的合成活跃,其合成速度升高,而在高浓度对硫磷的胁迫下,蛋白质,ＤＮＡ,ＲＮＡ的合成明显地受到了抑制,合成速度降低。