Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters

The present work reports on the daily cyclic variation of oxygen generation rates, carbon consumption rates, photosynthetic activities, growth rates and biochemical composition of the biomass in a pilot plant continuous outdoor culture of the microalgae Porphyridium cruentum. A linear relationship between the external irradiance and the average irradiance inside the culture was found. In addition, the oxygen generation and carbon consumption rates were found to be a function of the average irradiance inside the culture. A reduction in photosynthetic activity of the cells at noon and recovery in the afternoon was also observed. Therefore, the cells showed a short-term response of parameters such as oxygen generation rate as well as carbon consumption rate with external and average irradiance; a model of photosynthesis rate considering photoinhibition is proposed. This model is a useful tool for the operation and scaleup of tubular photobioreactors, and can be used for determining CO₂ requirements of the system. The higher the photosynthesis rates, the lower the carbon losses, ranging from 25% at noon to 100% during the night. The growth rate showed a linear relationship with the daily mean average irradiance inside the culture with a long-term response. Likewise, a linear relationship among the oxygen generation rate and the growth rate was obtained. With respect to the biochemical composition of the biomass, the cells showed a long-term response of metabolic routes to mean daily culture conditions. During the illuminated period, energy was stored as carbohydrates and synthesis of proteins was low. During the night, the stored carbohydrates were consumed. The fatty acid dry weight (DW) content decreased during the daylight period, whereas the fatty acid profile, as total fatty acids, was a function of growth rate. A short-term variation of exopolysaccharides synthesis with solar irradiance was also observed, i.e. the higher the external irradiance the higher the excretion of exopolysaccharides as a protection against adverse culture conditions.     

Modelling of a continuous pilot photobioreactor for microalgae production

In this study, a model of a continuous pilot photobioreactor for microalgae production is proposed. Three aspects have been studied: the modelling of kinetic growth, the gas-liquid transfer and the hydrodynamics in the photobioreactor. The modelling of each aspect has been developed with the dynamic simulation software SpeedUp, after experimental studies, then validated step-by-step. The connection of these three aspects aims to predict and optimise biomass production of the pilot plant.     

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。     

Increased production of extracellular polysaccharide by Porphyridium cruentum immobilized in foam sheets

Large‐size plate bioreactors were used to compare the production of extracellular polysaccharide by the red microalga Porphyridium cruentum when grown in suspension and in a foam sheet. A well‐defined illuminated area and unidirectional light propagation allowed us to generate information that is better quantified when expressed in terms of illuminated area. This is essential for meaningful comparison of data, especially considering that for a well‐designed and managed bioreactor, the culture production rates are believed to be light limited. At the same level of illumination, the culture immobilized in foam showed double the production rate of extracellular polysaccharide compared with the culture in suspension. The saturation level of biomass density per unit of illuminated area was eight times higher for the immobilized culture compared with the culture in suspension. Despite the increased biomass density for the immobilized culture, an increase in the light level above the optimum found for the culture in suspension reduced the extracellular polysaccharide production, suggesting that the photoinhibition light level was surpassed.     

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。     

Peroxidases from marine microalgae

Peroxidase activity was detected in cell-free extractsof strains of three species of the marine microalgae,Porphyridium purpureum, Phaeodactylumtricornutum and Dunaliella tertiolecta. However, no bromo- or chloroperoxidase activity wasdetected in any, using the standard 2-chlorodimedoneassay. Only the extract from P. purpureumoxidized iodide and this peroxidase was partiallypurified via anion-exchange chromatography. KI ando-dianisidine assay of the fractions indicatedthat only one peroxidase was present. Characterization of the thermally labile enzymesuggested that it is a heme-containing peroxidase,with a molecular weight of approximately 36,000.     

紫球藻藻红蛋白的分离纯化及光谱学特性

采用4℃反复浸提、离心、硫酸铵沉淀、DEAE—Sepharose Fast Flow离子交换柱层析,从紫球藻（Porphyridium cruentum Naegeli）冻干粉中分离纯化藻红蛋白,分离纯度达到4．85,总收率51．9％;经羟基磷灰石柱层析纯化,藻红蛋白纯度达到5．10,总收率34．0％,聚丙烯酰胺凝胶电泳显示1条带。所分离纯化的藻红蛋白含有3个亚基（α、β、γ）,在可见光区545nm和560nm处有2个吸收峰,在498nm处有1个吸收肩峰。实验结果说明所分离纯化的藻红蛋白纯度符合要求。     

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg⁻¹ fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.     

INTRASPECIFIC TRANSFER OF HERBICIDE RESISTANCE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYCEAE) VIA PROTOPLAST FUSION

Stable progeny doubly resistant to the herbicides sulfometuron methyl (SMM) and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (DCMU) were obtained at a frequency of 2% on fusion of protoplasts derived from mutants of Porphyridium sp. (UTEX 637) that were resistant only to SMM (strain SMR) or DCMU (strain DC-2). In the presence of both herbicides, only the fusion progeny could grow; both parental mutants were inhibited. In the absence of SMM, the activity of acetohydroxy acid synthase (AHAS) in the wild-type strain was similar to that in DC-2, exceeding that of SMR by up to 4.5-fold. AHAS activities of all fusion progeny were lower than those of the wild-type strain and DC-2 but higher than that of SMR. In the presence of SMM, AHAS activities of all tested fusion progeny ranged between those of the two parental mutants. This result indicates that both types of AHAS, the type resistant to SMM and the sensitive type, originating from SMR and DC-2, respectively, were expressed in the fusion progeny. In the presence of DCMU, the photosynthetic activity of SMR was completely inhibited, whereas that of DC-2 was unaffected. The photosynthetic activity of the fusion progeny in the presence of DCMU was slightly lower than that of DC-2. Both the cell volume and the DNA content of the fusion progeny were similar to those of the parents. However, the genetic nature of the fusion products has not yet been elucidated. To the best of our knowledge, this is the first report on transfer of herbicide resistance via protoplast fusion in algae.