Preliminary characterisation of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen

Haslea ostrearia is a common marine tychopelagic diatom which has the particularity of synthesizing a blue-green hydrosoluble pigment called “marennine”. This pigment, when released into the external medium, is known to be responsible for the colour of oyster gills. Here we present results for main biophysical and biochemical characteristics of pure intra- and extracellular marennine. Tests for chemical determination show that the nature of the two forms of marennine cannot be distinguished and could be related to a polyphenolic compound. Nevertheless, based on spectral properties and the molecular weight, which is about 10751 ± 1 and 9893 ± 1 Da, for the intracellular and extracellular forms respectively, we assess that the pigment accumulated in the apex of the cell and the one released in the external medium have probably distinct molecular structures.     

Effects of irradiance and growth phase on the ascorbic acid content of Isochrysis sp. T.ISO (Prymnesiophyta)

The effects of irradiance and growth phase on the concentration ofascorbic acid (AA) were examined in Isochrysis sp.(T.ISO),one of the most popular microalgal species used in aquaculture. Across fourdifferent irradiances (15, 40, 100 and 200 mol m^–2s^–1 with continuous illumination; i.e., lightconditionstypically found in hatcheries), the average % AA in cultures during logarithmicphase ranged from 0.38% (15 mol m^–2s^–1) to 0.49% (100 mol m^–2s^–1) of cell dry weight. Average % AA values forcultures at 40 and 100 mol m^–2s^–1 reduced by more than half to 0.16% and 0.20%,respectively with the onset of stationary phase.     

Flow cytometry for the development of biotechnological processes with microalgae

The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.     

CO<Subscript>2</Subscript> bio-mitigation using microalgae

Microalgae are a group of unicellular or simple multicellular photosynthetic microorganisms that can fix CO(2) efficiently from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts. Combination of CO(2) fixation, biofuel production, and wastewater treatment may provide a very promising alternative to current CO(2) mitigation strategies.     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Conditions for open-air outdoor culture of Dunaliella salina in southern Spain

The optimization of the operation, under the climatic conditions of southern Spain, of an experimental plant for -carotene production by Dunaliella has been pursued. The effects of mixing, culture depth, cell density and dilution cycles on -carotene and biomass productivity were studied under a semicontinuous culture regime in open tanks outdoors. Using 3 m²-surface containers, the highest productivity values, for both -carotene and biomass, were recorded with a flow rate of 0.55 m s^–1; 10 cm depth; 0.7 – 0.9 × 10⁶cell ml^–1, population density; and dilution cycles of two days. An average annual productivity of 1.65 g (dry wt) m^–2 d^–1 was estimated for Dunaliella biomass, being that for -carotene of about 0.1 g m^–2 d^–1. Under these optimized conditions, experiments have been carried out at the Cadiz Bay with 20 m²-surface tanks during a whole-year cycle. The results obtained have validated this location and the operating conditions established as being most appropriate for efficient mass production of -carotene rich D. salina.     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.     

Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils

This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. The lipase Novozym 435 (Novo Nordisk, A/S) from Candida antarctica was used to catalyze this reaction. The main factors influencing the degree of esterification and triglyceride yield were the amount of enzyme, water content, temperature and glycerol/fatty acid ratio. The optimum reaction conditions were established as: 100 mg of lipase; 9 ml hexane; 50°C; glycerol/PUFA concentrate molar ratio 1.2:3; 0% initial water; 1 g molecular sieves added at the start of reaction; and an agitation rate of 200 rpm. Under these conditions, a triglyceride yield of 93.5% was obtained from cod liver oil PUFA concentrate; the product contained 25.7% eicosapentaenoic acid and 44.7% docosahexaenoic acid. These optimized conditions were used to study esterification from a PUFA concentrate of the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. With the first, a triglyceride yield of 96.5%, without monoglycerides and very few diglycerides, was obtained after 72 h of reaction; the resulting triglycerides had 42.5% eicosapentaenoic acid. A triglyceride yield of 89.3% was obtained from a P. cruentum PUFA concentrate at 96 h of reaction, which contained 43.4% arachidonic acid and 45.6% EPA. These high triglyceride yields were also achieved when the esterification reaction was scaled up 5-fold.     

Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production

Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.     

The occurrence of the chloroplast pyrenoid is correlated with the activity of a CO2-concentrating mechanism and carbon isotope discrimination in lichens and bryophytes

The organic-matter carbon isotope discrimination () of lichens with a wide range of photobiont and/or cyanobiont associations was used to determine the presence or absence of a carbon-concentrating mechanism (CCM). Two groups were identified within the lichens with green algal photobionts. One group was characterised by low, more C₄-like values ( < 15), the other by higher, more C₃-like values ( > 18). Tri-partite lichens (lichens with a green alga as the primary photobiont and cyanobacteria within internal or external cephalodia) occurred in both groups. All lichens with cyanobacterial photobionts had low values ( < 15). The activity of the CCM, organic-matter values, on-line values and gas-exchange characteristics correlated with the presence of a pyrenoid in the algal chloroplast. Consistent with previous findings, lichens with Trebouxia as the primary photobiont possessed an active CCM while those containing Coccomyxa did not. Organic values for lichens with Stichococcus as the photobiont varied between 11 and 28. The lichen genera Endocarpon and Dermatocarpon (Stichococcus + pyrenoid) had C₄-like organic values ( = 11 to 16.5) whereas the genus Chaenotheca (Stichococcus — pyrenoid) was characterised by high C₃-like values ( = 22 to 28), unless it associated with Trebouxia ( = 16). Gas-exchange measurements demonstrated that Dermatocarpon had an affinity for CO₂ comparable to those species which possessed the CCM, with K_0.5 = 200–215 1 · 1^–1, compensation point () = 45–48 l · l^–1, compared with K_0.5 = 195 1 · 1^–1, = 441 · 1^–1 for Trebouxioid lichens. Furthermore, lichens with Stichococcus as their photobiont released a small pool (24.2 ± 1.9 to 34.2 ± 2.5 nmol · mg^–1 Chl) of inorganic carbon similar to that released by Trebouxioid lichens [CCM present, dissolved inorganic carbon (DIC) pool size = 51.0 ± 2.8 nmol · mg^–1 Chl]. Lichens with Trentepohlia as photobiont did not possess an active CCM, with high C₃-like organic values ( = 18 to 23). In particular, Roccella phycopsis had very high on-line values ( = 30 to 33), a low affinity for CO₂ (K_0.5 = 400 1 · 1^–1, = 120 1 · ^–1) and a negligible DIC pool. These responses were comparable to those from lichens with Coccomyxa as the primary photobiont with Nostoc in cephalodia (organic = 17 to 25, on-line = 16 to 21, k_0.5 = 388 1 · 1^–1, = 85 1 · 1^–1, DIC pool size = 8.5 ± 2.4 nmol · mg^–1 Chl). The relative importance of refixation of respiratory CO₂ and variations in source isotope signature were considered to account for any variation between on-line and organic . Organic was also measured for species of Anthocerotae and Hepaticae which contain pyrenoids and/or Nostoc enclosed within the thallus. The results of this screening showed that the pyrenoid is correlated with low, more C₄-like organic values ( = 7 to 12 for members of the Anthocerotae with a pyrenoid compared with = 17 to 28 for the Hepaticae with and without Nostoc in vesicles) and confirms that the pyrenoid plays a fundamental role in the functioning of the CCM in microalgal photobionts and some bryophytes.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIC dissolved inorganic carbon (CO₂ + HCO ₃ ^- + CO ₃ ^2- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - photobiont photosynthetic organism present in the lichen - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This research was funded by Natural Environment Research Council grant no. GR3/8313. The authors would also like to thank Dr. B. Coppins, Royal Botanic Gardens Edinburgh and Prof. A. Roy Perry, National Museum of Wales, for access to herbarium collections, Dr. T. Booth for confocal microscopy work and Dr. A.J. Richards, University of Newcastle upon Tyne and Dr. O.L. Gilbert, University of Sheffield for identifying bryophytes and lichens respectively. E.S. would particularly like to thank Dr. M. Broadmeadow, The Forestry Authority, Farnham, Surrey, and Cristina Máguas, Universidade de Lisboa, for their advice and expertise at the beginning of the project.