In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

Summary Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.     

Hydrodynamic characteristics and microalgae cultivation in a novel flat‐plate photobioreactor

Flat‐plate photobioreactors (FPPBRs) are widely reported for cultivation of microalgae. In this work, a novel FPPBR mounted with inclined baffles was developed, which can make the fluid produce a “spirality” flow. The flow field and cell trajectory in the photobioreactor were investigated by using computational fluid dynamics. In addition, the cell trajectory was analyzed using a Fast Fourier transformation. The influence of height of the baffles, the angle α between the inclined baffle and fluid inlet flow direction (z), and the fluid inlet velocity on the frequency of flashing light effect and pressure drop were examined to optimize the structure parameters of the inclined baffles and operating conditions of the photobioreactor. The results showed that with inclined baffles built‐in, significant swirl flow could be generated in the FPPBR. In this way, the flashing light effect for microalgal cell could also be achieved and the photosynthesis efficiency of microalgae could be promoted. In outdoor cultivation of freshwater Chlorella sp., the maximum biomass productivity of Chlorella sp. cultivated in the photobioreactor with inclined baffles was 29.94% higher than that of the photobioreactor without inclined baffles. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

The profiles of nitrate reductase and carbonic anhydrase activity in batch cultivation of the marine microalgae Tetraselmis gracilis growing under different aeration conditions

Tetraselmis gracilis, a Prasinophycean alga found in estuaries and in the open ocean, was cultivated under different conditions of aeration, which resulted in variations of inorganic carbon in the medium. Relative growth rates, nitrate reductase and carbonic anhydrase activities were daily determined and correlated to the concentration of nitrate, nitrite, phosphate, inorganic and organic carbon in the media. Nitrate reductase catalyzes the reversible carbon dioxide hydration reaction. The activity profiles of both enzymes during 10 days of cultivation under aeration with air showed an inverse relationship: the maximum in the activity of nitrate reductase coincided with the minimum of carbonic anhydrase activity. An ionizable organic carbon species with pKa in the range of metabolites of the photorespiratory path was found parallel with the increase of carbonic anhydrase activity and the decrease of nitrate reductase activity. The onset of photorespiration is probably one of the factors involved in the simultaneous regulation of these enzymatic processes. Cultures aerated with air containing 5% CO2 showed different profiles for nitrate reductase activity and nitrate uptake.     

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.     

Application of a growth-promoting bacteria for stable mass culture of three marine microalgae

The practical mass culture of marine microalgae, facesoccasionally unexpected problems or collapse. The effect of amarine bacterium, Flavobacterium sp., which was found topromote growth of a marine diatom Chaetoceros gracilisin the axenic culture condition, was examined on the masscultures of three marine microalgae.Three marine microalgae (C. gracilis, Isochrysisgalbana, and Pavlova lutheri) were mass cultured in 3 lflatbottom flasks (2.5 l capacity of culture medium), in anindoor culture room at a commercial pearl oyster hatchery. Themicroalgal cells and the bacterium were inoculated at the sametime, in the culture media. The specific growth rate andmaximal cell density were determined in treated cultures (withadded bacterial strain) and in controls (without addedbacterial strain). The specific growth rate of C.gracilis in treated cultures was significantly higher thanthat of control cultures, and the stationary growth phase inthe treated cultures lasted longer till the end of the cultureperiod. However, the bacterium had no apparent effect on theexponential growth phase of two phytoflagellates, I.galbana and P. lutheri, but kept longer the high celldensity in the stationary growth phases. The added bacterialstrain (Flavobacterium sp.) was the dominant species(more than 45%) among the bacterial flora during the cultureperiod.     

Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach

Abstract

Optimizing the production of the high-value renewables such as OMEGAs through pathway engineering requires an in-depth understanding of the structure–function relationship of genes involved in the OMEGA biosynthetic pathways. In this preliminary study, our rationale is to identify and characterize the ～221 putative genes involved in production of OMEGAs using bioinformatic analysis from the Streptophyte (plants), Chlorophyte (green algae), Rhodophyta (red algae), and Bacillariophyta (diatoms) lineages based on their phylogenomic profiling, conserved motif/domain organization and physico-chemical properties. The MEME suite predicted 12 distinct protein domains, which are conserved among these putative genes. The phylogenomic analysis of the putative candidate genes [such as FAD2 (delta-12 desaturase); ECR (enoyl-CoA reductase); FAD2 (delta-12 desaturase); ACOT (acyl CoA thioesterase); ECH (enoyl-CoA hydratase); and ACAT (acetyl-CoA acyltransferase)] with similar domains and motif patterns were remarkably well conserved. Furthermore, the subcellular network prediction of OMEGA biosynthetic pathway genes revealed a unique interaction between the light-dependent chlorophyll biosynthesis and glycerol-3-phosphate dehydrogenase, which predicts a major cross-talk between the key essential pathways. Such bioinformatic analysis will provide insights in finding the key regulatory genes to optimize the productivity of OMEGAs in microalgal cell factories.     

Culture of human umbilical vein endothelial cells using 96-well microplates and position effects on cell growth

When endothelial cells isolated from human umbilical veins were cultured for 6 days using 96-well microplates, the final cell density in each well was found not to be the same although the medium composition of each well was exactly the same. Cell growth in the wells located at the periphery of a microplate was low, while that in the central area of the plate was high. A possible cause for different rate of growth was proposed as the uneven concentration of oxygen in the culture medium of each well.     

Expression of soluble recombinant proteins in a cell-free system using a 96-well format

For structural and functional genomics programs, new high-throughput methods to obtain well-expressing and highly soluble proteins are essential. Here, we describe a rapid procedure to express recombinant proteins in an Escherichia coli cell-free system using a 96-well format. The identification of soluble proteins is performed by the Dot Blot procedure using an anti-His tag antibody. The applications and the automation of this method are described.     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

Variation in some photosynthetic characteristics of microalgae cultured in outdoor thin-layered sloping reactors

In outdoor thin-layer sloping reactors algae are batch cultured and harvested at biomass concentrations of about 15 g (dw) I^-1 whereafter a portion is used as inoculum for the next cycle. Light saturation curves of the oxygen evolution (PII curves) of the algae were measured using diluted aliquots of suspension taken from the reactors. The maximum specific photosynthetic rates (P ^B _max) and the light intensity at the onset of saturated photosynthesis (I _k ) decreased whilst the maximum specific photosynthetic efficiency ( ^B ) increased with an increase in the biomass concentration, during the production cycle. These differences reflect transition from light- to dark-acclimated state of the algae that occurs as a result of an increase of the suspension concentration during the production cycle. During these experiments the thin-layered smooth sloping cultures (TLSS, culture depth 5–7 mm) had higher photosynthetic rates per volume than the thin-layered baffled sloping cultures (TLBS, culture depth 5–15 mm). This was ascribed to the higherP ^B _max values of the algae grown in the TLSS cultures, allowing them to utilise high incident irradiancies more effectively. However, the areal productivity of the TLBS was higher than the TLSS indicating a higher photosynthetic efficiency of the TLBS reactors. The specific productivity decreased rapidly with an increase in the biomass concentration, but the yield remained linear during the batch production cycle, even at high areal densities.     

The extracellular polysaccharides of the red microalgae: Chemistry and rheology

Cells of red microalgae encapsulated within sulphated polysaccharides, are thought to have a wide range of potential industrial applications. Our group is thus carrying out a comprehensive research program aimed at bringing these biopolymers into industrial use. The program includes physiological studies on polysaccharide production, outdoor cultivation of the microalgae, and characterisation of the polysaccharides. Chemical composition and structure and physicochemical properties were investigated for the polysaccharides of three red microalgae, Porphyridium sp., P. aerugineum and Rhodella reticulata. Differences were found among the three species in the composition of the monosugars, half ester sulphate groups and glucuronic acid content, but a disaccharide isolated was identical in all the species examined. This disaccharide is thought to be the basic building block of these polysaccharides. In addition, monosugar sulphates were isolated and characterised. Fractionation by charge showed the polysaccharides to be heterogenous and composed of at least two fractions that differed in their composition. Although the polysaccharides differed in composition, their rheological characteristics were found to be similar. Aqueous solutions of the biopolymers were stable over a wide range of pH values and temperatures and were compatible with monovalent cations. Mixtures of the algal polysaccharides with locust bean gum exhibited synergism and syneresis. When the gel strength was compared with that of agar gel at the same concentration the polysaccharide gels were found to be weaker.     

成都地区天然水域中异养微藻的分离及其特性研究

采用添加不同浓度和种类抑菌剂的平板进行多次平板划线分离和三角瓶培养,从成都地区四川大学望江及江安校区附近水域中分离得到了一株可在无光照条件下异养生长的微藻,经初步鉴定为小球藻并编号为Chlorella SCU-01.在此基础上初步研究了该藻株在不同葡萄糖起始浓度下的异养生长及色素合成特性.     

The role of microalgae in aquaculture: situation and trends

Algae are utilized diversely in aquaculture, but theirmain applications are related to nutrition. They areused in toto, as a sole component or as a foodadditive to supply basic nutrients, color the flesh ofsalmonids or for other biological activities. The needfor nutritional sources safer than traditional animalproducts has renewed interest in plants in general andalgae in particular. This report deals principallywith the nutritional role of microalgae inaquaculture.The larvae of molluscs, echinoderms andcrustaceans as well as the live prey of some fishlarvae feed on microalgae. Though attempts have beenmade to substitute inert particles for thesemicro-organisms which are difficult to produce,concentrate and store, only shrimp and live prey forfish will accept inert food, and only shrimp accept itfully. Several studies have confirmed that a live,multi-specific, low-bacteria microalgal biomassremains essential for shellfish hatcheries. Majoradvances are expected from new production systemdesigns and operations, from batch-run open tanks tomore sophisticated continuously run and closed loopreactors. Studies are underway to simplify hatcheryoperations by replacing biomass produced on-site withrun-times by that produced and preserved elsewhere.Although still promising, they have not given rise, sofar, to any application for molluscs. Otherapplications of microalgae in aquaculture, from greenwater to making salmon flesh pinker, are examined.Whether produced on or off-site, there remains thequestion of cost effectiveness of microalgalproduction systems. This can only be achieved bysubstantial upscaling and improved quality control.     

Recent progress in the use of processed microalgae in aquaculture

Mass-cultured algal biomass has been tested as a food source for a number of aquaculture animals because of its low cost and convenience. This paper reviews the results of nutritional studies on processed microalgae with respect to mollusc, crustacean, rotifer and fish culture. Research using species of Spirulina, Chlorella, Scenedesmus and other mass-produced algae indicates that microalgae can be an effective dietary component provided that processing, diet formulation and presentation requirements are met. Processed microalgae can be used to correct specific dietary deficiencies in artificial diets. Our research found that the growth and pigmentation of marron, Cherax tenuimanus (Decapoda, Crustacea), can be significantly enhanced by the incorporation of Dunaliella salina in its artificial diet. Likewise, rainbow trout, Oncorhynchus mykiss, were pigmented by Haematococcus pluvialis.     

The production of clonal and axenic cultures of microalgae using fluorescence-activated cell sorting

Unialgal cultures of the flagellate algae Cyanophora paradoxa, Haematococcus lacustris, Monomastix sp., Scherffelia dubia and Spermatozopsis similis which contained bacteria were sorted by flow cytometry to obtain axenic clonal cultures. The variables used for fluorescence-activated cell sorting (FACS) were chlorophyll autofluorescence, forward scatter and side scatter of the laser beam. To produce clonal cultures, a single cell was sorted into each culture flask. Depending on the species, about 20–30% of the sorted cultures grew successfully and at least 20% of these were axenic even if the numerical ratio betweeen bacteria and algae in the original cultures was as high as 300:1. FACS represents an effective and rapid method for the preparation of clonal and axenic cultures of microalgae.     

A strategy for high cell density culture of heterotrophic microalgae with inhibitory substrates

Substrate inhibition is one of the major problems preventing high cell densities of microalgae in heterotrophic culture, so the possibility of overcoming the problem by various culture techniques was examined. It was found that perfusion culture may be the most appropriate technique for high cell densities in heterotrophic culture using inhibitory substrates. An experimental example in which a hollow fibre cell recycle system (HFCRS) was employed to achieve high cell densities of Chlamydomonas reinhardtii on acetate under heterotrophic conditions of growth was demonstrated. The cell density in the HFCRS was much higher than that reported in the literature for this species.     

A fluidised bed vessel for the culture of immobilised plant cells and its application for the continuous production of fine cell suspensions

With the expansion of immobilised plant cell technology the need has arisen for a suitable vessel in which systems can be efficiently manipulated.Described is a vessel which incorporates many of the features of a fluidised bed, together with some of those from airlift technology to enable immobilised plant cells to be cultured at high biomass concentrations while maintaining a high mass transfer and controlled aeration under continuous flow conditions. In the case described, the vessel has been used for the continuous production of fine plant cell suspensions, although it is easily adaptable for use in cell mediated biotransformation or de novo synthesis studies.Abbreviations 2,4D 2.4 dichlorophenoxyacetic acid