El-Hassan Belarbi

RECOVERY OF MICROALGAL BIOMASS AND METABOLITES: PROCESS OPTIONS AND ECONOMICS

Commercial production of intracellular microalgal metabolites requires the following: (1) large-scale monoseptic production of the appropriate microalgal biomass; (2) recovery of the biomass from a relatively dilute broth; (3) extraction of the metabolite from the biomass; and (4) purification of the crude extract. This review examines the options available for recovery of the biomass and the intracellular metabolites from the biomass. Economics of monoseptic production of microalgae in photobioreactors and the downstream recovery of metabolites are discussed using eicosapentaenoic acid (EPA) recovery as a representative case study.

Rapid simultaneous lipid extraction and transesterification for fatty acid analyses

An improved adaptation of the direct transesterification method of Lepage and Roy (J. Lipid Res. 25, 1391–96, 1984) for the preparation of fatty acid methyl esters allows notable saving of time and reagents. The material being analysed is heated for 10 minutes with methanol, acetyl chloride and hexane.

Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum

The influence of culture age and nitrogen concentration on the distribution of fatty acids among the different acyl lipid classes has been studied in continuous cultures of the microalga Phaeodactylum tricornutum. The culture age was tested in the range of 1.15-7 days, controlled by adjusting the dilution rate of fresh medium supplied. The effect of nitrogen concentration was tested from saturating conditions to starvation by modifying nitrate concentration in the fresh medium. Culture age had almost no influence on the fatty acid content; 16:0, 16:3 and 20:5 increased moderately wherein the level of 16:1 decreased when the culture age decreased. Culture age had no effect on the total fatty acid content that remained around 11% of dry weight. Conversely, culture age had a greater impact on lipid classes, producing changes in amounts of triacylglycerols (TAG) which ranged between 43% and 69%, and galactolipids (GLs) that oscillated between 20% and 40%. In general, the content of polar lipids of the biomass decreased with culture age. The other factor assayed, nitrogen content, affected the fatty acid profile. Saturated and monounsaturated fatty acids accumulated when the nitrogen concentration was decreased. The experiments regarding the effect of nitrogen concentration on lipid species were carried out with cells of an average age of 3.5 days. A decrease of the nitrogen concentration caused the GL fraction to decrease from 21 to 12%. Conversely, both neutral lipids (NLs) and phospolipids (PLs) increased from about 73 to 79% and from 6 to 8%, respectively. In these experiments, TAG was the lipid class with the highest increase, from 69 to 75%.

Acyl lipids of three microalgae

Abstract Acyl-lipid composition of Isochrysis galbana, Porphyridium cruentum and Phaeodactylum tricornutum from stationary-phase cultures has been analyzed by TLC and GC. Additionally, P. tricornutum from an outdoor tubular photobioreactor was also studied. Neutral (NLs) and glyco (GLs) lipids were found in similar amounts of ca 40–45% each, with phospholipids (PLs) representing ca 10–20%. The major lipid classes were triacylglycerol (TAG), monogalactosylaylglycerols (MGD), and galactosylacylglycerols (DGD), usually in that order. The P. tricornutum biomass taken from the bioreactor showed a distinctive acyl-lipid composition. GL content was nearly twice (56%) that of the indoor culture (31%) and NL content (31%) was nearly half that of the indoor culture (54%). These changes mainly involved TAG and MGD. The fatty acid composition of lipids in the outdoor culture generally remained unaffected, except for MGD, DGD, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in which eicosapentaenoic acid (EPA) content was greatly increased. TAG, the main class of lipids, always contained a high proportion of EPA, 16:0 and 16:1. In the typical (SQD) composition, 14:0, 16:0 and 16:1 accounted for around 70% of fatty acids, with small amounts of polunsaturated fatty acids (PUFAs). The lipid classes which typically had the highest PUFA content in the microalgae were MGD and DGD. In P. tricornutum , these lipids characteristically contained a large proportion of 16:1, 16:2, 16:3 and 16:4. PC, which was composed of the main C 16 fatty acids and the major PUFAs, was usually the most abundant phospholipid. Phosphatidylgycerol showed an accumulation of 16:1 (probably a mixture of 16:1n7 and 16:1n3 trans ) as a distinctive feature. Phosphatidylinositol was characterized by 16:0 and 16:1 (and 14:0 in I. galbana ), which together accounted for over 50% of fatty acids, and significant presence of the main PUFAs. There was no consistent fatty acid pattern for PE in the three microalgae studied. PE was exceptional in I. galbana , containing 61% docosahexaenoic acid.

γ-Linolenic acid purification from seed oil sources by argentated silica gel chromatography column

Abstract The polyunsaturated fatty acid γ-linolenic acid (GLA,18:3ω6), which has several pharmaceutical properties, has been purified from the seed oil of three plant species, Anchusa azurea, Scrophularia sciophila and Echium fastuosum. The process consists of four main steps, (i) simultaneous extraction and saponification of the seeds; (ii) urea fractionation method; (iii) urea-concentrate methylation; (iv) argentated silica gel column chromatography of the urea-concentrate methylated. Argentated silica gel chromatography yields high purity GLA in the process in A. azurea and S. sciophila, with yields in the combined process of 73 and 64%, respectively. With E. fastuosum, the recovery for the combined process was 60%, with a final purity 86%, due to the presence of the α-linolenic acid (ALA, 18:3ω3), which elutes with GLA in the chromatography column. The suitability of a process without urea concentration is also compared and discussed.

RETRACTED: A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

This article has been retracted at the request of the Editor. Please see Elsevier Policy on Article Withdrawal ( http://www.elsevier.com/locate/withdrawalpolicy ). Reason: It duplicates significant parts of a paper that has already been published by the same authors in Enzyme Microb. Technol., volume 26 (2000) 516–529, doi: 10.1016/S0141-0229(99)00191-X . One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper is not under consideration for publication elsewhere. The scientific community takes a very strong view on this matter and we apologize to readers of the journal that this was not detected during the submission process.

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640.

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L‐15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L−1) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopusaqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long‐term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.

Hexane reduces peroxidation of fatty acids during storage

The free fatty acids eicosapentaenoic acid (C20:5ω3, EPA) and docosahexaenoic acid (C22:6ω3), obtained from the microalgae Phaeodactylum tricornutum, and the EPA methyl ester were compared with regard to their extent of peroxidation using different storage conditions. Several series were stored according to selected variables: hexane addition versus no addition, 4 °C versus 25 °C, and antioxidant addition (octyl gallate) versus no antioxidant addition, always in the dark. Previously, the EPA and methyl EPA structures were confirmed by NMR spectra to discard the formation of conjugated dienes after the downstream process. The results showed that the stability was higher for methyl EPA than for the free fatty acid, and that peroxidation can be retarded by low temperature storage and mainly by hexane addition. The peroxidation process was evaluated by the peroxide value (spectrophotometric method by iodine absorption), although the conjugated diene absorbance and the loss in percentage of the fatty acid have been tested as good indicators of the peroxidation process. A simple kinetic model that explains the peroxidation process during the initiation and propagation steps is given.

A bioreaction-diffusion model for growth of marine sponge explants in bioreactors

Marine sponges are sources of high-value bioactives. Engineering aspects of in vitro culture of sponges from cuttings (explants) are poorly understood. This work develops a diffusion-controlled growth model for sponge explants. The model assumes that the explant growth is controlled by diffusive transport of at least some nutrients from the surrounding medium into the explant that generally has a poorly developed aquiferous system for internal irrigation during early stages of growth. Growth is assumed to obey Monod-type kinetics. The model is shown to satisfactorily explain the measured growth behavior of the marine sponge Crambe crambe in two different growth media. In addition, the model is generally consistent with published data for growth of explants of the sponges Disidea avara and Hemimycale columella. The model predicted that nutrient concentration profiles for nutrients, such as dissolved oxygen within the explant, are consistent with data published by independent researchers. In view of the proposed model’s ability to explain available data for growth of several species of sponge explants, diffusive transport does play a controlling role in explant growth at least until a fully developed aquiferous system has become established. According to the model and experimental observations, the instantaneous growth rate depends on the size of the explant and all those factors that influence the diffusion of critical nutrients within the explant. Growth follows a hyperbolic profile that is consistent with the Monod kinetics.