Continuous ‐ discrete interval observers for monitoring microalgae cultures

Algorithms estimating unmeasured component concentrations play a key role in bioprocess applications where only a few on‐line measurements are usually available. In this article, interval observers are designed to provide guaranteed intervals for the key components involved in cultures of microalgae. In contrast with most of the published studies focusing on continuous‐time measurements, this study considers discrete‐time measurements with possibly long and irregular sampling and defines predictors based on model equations and state transformations to ensure the enclosure of the state variables between two measurement times. The methods are validated with experimental data where the remaining inorganic nitrogen and the microalgal internal quota are estimated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Changes in the CO2 Concentrating Mechanism During the Cell Cycle in Dunaliella tertiolecta

Synchronised cells of Dunaliella tertiolecta were used to investigate the expression of the CO₂ concentrating mechanism over the cell cycle during growth in either ambient air (low Ci cells) or air enriched with 5% CO₂ (high Ci cells). The cultures were analysed for extracellular carbonic anhydrase activity, affinity of photosynthesis for inorganic carbon (Ci) and the ability to accumulate Ci. In high Ci cells, carbonic anhydrase activity changed between 2 ? 4 units mg^?1 Chi during the light-dark rhythm showing no clear periodicity. Similarly, the apparent affinity for Ci remained rather constant over the cell cycle. This was judged from the Ci concentrations required for half maximum rate of photosynthesis (K_1/2(Ci)) of 72 ? 80μM. In the same cells the accumulation ratio of internal Ci versus external Ci ranged between 5 and 9.5 without a clear rhythm. In contrast, these parameters showed distinct periodical changes in synchronised low Ci cells. Carbonic anhydrase activity changed from 10 to 350 units mg^?1 Chl with maximum and minimum activities occurring in the middle and at the end of the light period, respectively. The K_1/2(Ci) values showed similar periodicity ranging between 13 ? 36μM. In addition the accumulation ratio increased up to 30 in the middle of illumination and decreased to its lowest level of 12 at the end of the light period. These results indicate the presence of a common step in regulating the induction of the measured parameters and that light is not an absolute requirement for the induction of the CO₂ concentrating mechanism in synchronous low CO₂ grown cells of Dunaliella tertiolecta.     

Fine structure of photo-kinetic systems in Dinobryon cylindricum var. Alpinum (chrysophyceae)

The ultrastructure of the stigma and associated flagellar-microtubular systems in Dinobryon cylindricum var. alpinum is described in detail and compared with observations on comparable photo-kinetic systems in other chrysophycean organisms. The chloroplastidic stigma of D. cylindricum var. alpinum is shown to lie in a particular positional relationship to the flagellar swelling in the anterior furrow and to several other organelles, to consist of a monolayer of c. 40 pigmented granules, each c. 250–500 nm diameter, arranged in a definite pattern, and to be overlain by several membrane systems. Other cytoplasmic pigmented bodies with dense crystalline contents surrounded by a single “unit membrane” aggregate near the anterior furrow on the side opposite the stigma. The swelling on the proximal portion of the smooth flagellum is separated from the plasmalemma of the anterior furrow by a nearly constant distance of 75–100 nm, has a multilamellate substructure that is linked by fine radiating interconnections to the axoneme doublets, and is connected to the plasmalemma by a system of fibrillar interconnections. A transitional helix in the basal body region is described as similar to structures reported in other chrysophycean flagellates. A striated rhizoplast with a periodicity of c. 90 nm extends from basal body I to the nuclear envelope. A seven-stranded microtubular root extends from the same basal body. Other fibrous and microtubular root systems are also described. The inter-relationships and possible functions of the aforementioned structures are discussed.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

Ultrastructure of the Marine Cryptomonad Chroomonas salina Cultured under Conditions of Photoautotrophy and Glycerol-Heterotrophy*

SYNOPSIS. Chroomonas salina was cultured in seawater medium enriched with nitrate, phosphate, silicate, trace-metal ions, and vitamins, under 3 conditions: (A) light without other organic additions (photoautotrophic); (B) light and added glycerol (photoheterotrophic); (C) in darkness but with added glycerol (chemoheterotrophic). The heterotrophic cultures were initiated from a stock maintained on glycerol in continuous darkness for 41/4 years. The autotrophic culture was initiated from a corresponding stock maintained under continuous illumination without any organic growth substrate. The fine structure of organisms from simultaneously initiated cultures was compared after 1, 2, 3, and 4 weeks of growth. “Young'’cells from the autotrophic and heterotrophic cultures of comparable maturity had no recognizable ultrastructural difference. In organisms from both the photoautotrophic and photoheterotrophic cultures there was a progressive accumulation of starch and lipid with aging, but whereas in cells from the former the production of starch was arrested after early growth and lipid was concentrated thereafter, in those from the latter both metabolites continued to be produced with consequent rapid degeneration of the cytoplasm followed by autolysis. By contrast, flagellates grown in the chemoheterotrophic culture accumulated only starch, with vacuole formation replacing the lipid stores. In all cases, the lipid bodies appeared to differ from the membrane-bound droplets normally observed, which actually diminished with aging. Starch accumulation appeared to cause more rapid cytopathologic changes and autolysis. No evidence of chloroplastic phycobilisome-type aggregations was noted in organisms from any culture at any age.     

短小芽孢杆菌对盐藻SZ-05的生物量和β-胡萝卜素产量的影响

研究了短小芽孢杆菌（Bacillus pumilus）对盐藻空间诱变株系SZ-05（Dunaliella salina SZ-05）的生物量及β-胡萝卜素积累的影响。结果表明,短小芽孢杆菌显著提高了盐藻SZ-05的生物量和β-胡萝卜素的产量,明显降低了培养体系中的溶解氧和胞外多糖的含量。溶解氧的减少,使得藻细胞的光呼吸作用下降,光合作用速率提高,使藻细胞生物量增加。胞外多糖具有抗氧化作用,胞外多糖的减少可能进一步增加了β-胡萝卜素的合成,从而使β-胡萝卜素在胁迫条件下大幅度增加。     

Pilot culture of three strains of Dunaliella salina for β-carotene production in open ponds in the central region of Iran

Summary Three strains of Dunaliella salina (I, G and A) were cultivated under the climatic conditions of Iran, in open ponds to compare the β-carotene production and the specific rate of growth. The experiments were accomplished in two separate stages. In the first stage, the cells were grown in ponds on nutrient-rich medium containing 2 M NaCl to obtain the necessary biomass. In the second stage, cells were stressed on nutrient-poor medium containing 2.5 M NaCl for β-carotene induction. The results showed that the specific growth rate of strain I was the highest during the first stage, whereas during the second stage, the growth rates of three strains were approximately the same. The overall results indicated that strain G had the highest potential for β-carotene accumulation of the strains tested and hence it was concluded that this strain is more suitable for outdoor cultivation under the climatic conditions of Iran than the other two.     

Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.