System design for the autotrophic production of microalgae

The mass culture of microalgae has been explored over the past 35 years for the production of food, fuels and chemicals, as well as for waste treatment and gas exchange. Culture systems have evolved over this period from small experimental systems to the commercial systems which are presently in place for treatment of municipal wastewaters and for production of a number of specialty products. Further design innovation and refinement will need to draw heavily on the experience which has been gained through the operation of systems employing a wide variety of designs.     

Nutritional properties of four marine microalgae for albino rats

The nutritive value of the marine microalgaeTetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta andChlorella stigmatophora was studied in diets given to rats. Biological assays were carried out in order to determine the Protein Efficiency Ratio (PER) and the Food Conversion Efficiency (FCE). Each dried microalga was fed to weaning Wistar albino rats as the sole protein source at a protein level of 12%. Control rats were given diets containing 12 % casein. Food consumption was similar in all groups. PER values obtained were 1.14 withT. suecica diet, 1.13 withI. galbana diet, 2.07 withD. tertiolecta diet and 1.13 withC. stigmatophora diet (casein, 2.50). FCE values followed a similar pattern. The data showed that the marine microalgaD. tertiolecta is a source of protein of good quality. Its PER is quite high, compared to vegetable and cereal proteins, and compares favourably with other microbial protein sources, such as yeasts or different freshwater microalgae. Haematological tests showed no significant differences among the groups in haemoglobin levels, red and white blood cell counts, differential count and mean corpuscular volume. Different blood parameters were also determined and a significant decrease in triglycerides levels appeared with all the microalgal diets, whereas a significant decrease in cholesterol appeared inD. tertiolecta andC. stigmatophora diets.     

Biotechnology of algal biomass production: a review of systems for outdoor mass culture

Microalgae are very efficient solar energy converters and they can produce a great variety of metabolites. Man has always tried to take advantage of these proporties through algal mass culture. Despite the fact that many applications for microalgae have been described in the literature, these micro-organisms are still of minor economic importance. Industrial reactors for algal culture are at present, all designed as open race-ways (shallow open ponds where culture is circulated by a paddle-wheel). Technical and biological limitations of these open systems have given rise to the development of enclosed photoreactors (made of transparent tubes, sleeves or containers and where light source may be natural or artificial). The present review surveys advances in these two technologies for cultivation of microalgae. Starting from published results, the advantages and disadvantages of open systems and closed photobioreactors are discussed. A few open systems are presented for which particularly reliable results are available. Emphasis is then put on closed systems, which have been considered as capital intensive and are justified only when a fine chemical is to be produced.     

Total fatty acid composition as a taxonomic index of some marine microalgae used as food in marine aquaculture

Total lipid and total fatty acid composition was studied in twelve marine microalgae species, belonging to the Eustigmatophyceae, Chlorophyceae, Haptophyceae and Prasinophyceae, cultured under comparable conditions.Species of the same class showed a particular fatty acid composition, indicating specific fatty acid bioconversion modes. The fatty acid composition of the different microalgae was compared to previously published data.     

A laboratory-scale system for mass culture of freshwater microalgae in polyethylene bags

A laboratory-scale system for mass culture of microalgae in 8-, 20- and 40-L polyethylene bags, was designed. Bags are 16.8 cm diameter and 52 cm (8-L bags), 112 cm (20-L) or 224 cm (40-L) length. The system was tested successfully with two freshwater microalgae,Ankistrodesmus falcatus andScenedesmus incrassatulus, cultured in Bold's Basal medium (prepared with either deionized or tap water). The procedure described is simple, reliable and practical, and enables a very cost-effective production of freshwater microalgae to satisfy any laboratory requirements, and when quantities demanded for special applications can not be met by the standard laboratory culture procedures.     

An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae

Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical methods employed. The different extraction procedures (exposure to water, grinding, etc.), protein precipitation using different amounts of 25% trichloroacetic acid and quantification of protein by two different methods and using two protein standards were evaluated. All procedures were tested using freeze-dried samples of three macroalgae: Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocol for protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure water for 12 h, followed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should be done with 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in this study was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima, Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae (Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Comparison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry's method should be used instead of Bradford's using bovine serum albumin (BSA) as protein standard instead of casein. This may be related to the reactivity of the protein standards and the greater similarity in the amino acid composition of BSA and algae. The current results should contribute to more accurate protein determinations in marine algae.     

Cadmium adsorption by the non-living biomass of microalgae grown in axenic mass culture

The freeze-dried (extracted and non-extracted) biomass of 15 microalgal species grown in axenic mass culture and belonging to the Cyanobacteria, Chloro-, Eustigmato-, Phaeo-, Rhodo- and Tribophyceae were investigated for their ability to adsorb cadmium (Cd) ions from aqueous solutions. For comparison, other standard adsorbing materials (activated carbon, silica gel, siliceous earth) were included in the studies. The biomass of 11 microalgae exhibited a higher Cd adsorption than the standard materials. Extraction of the algal biomass increased the Cd adsorption capability of some, but not all microalgae. High Cd adsorption was found inAnabaena lutea, Nodularia harveyana, andNostoc commune (Cyanobacteria),Chlamydomonas sp. (Chlorophyceae),Bumilleriopsis filiformis (Tribophyceae), and inEctocarpus siliculosus, Halopteris scoparia andSpermatochnus paradoxus (Phaeophyceae). The specific surface (m² cm^–3) of the various microalgae was determined by means of laser diffractometry.Anabaena inaequalis andA. lutea (Cyanobacteria) and the Phaeophyceae had especially high Cd adsorption per surface unit. Most of the Cd adsorbed to these various materials could be desorbed subsequently with diluted mineral acid (pH 2).     

Screening of marine microalgae for bioremediation of cadmium-polluted seawater

Twenty four strains out of 191 marine microalgal strains exhibited cadmium (Cd) resistance. They were tested for their Cd removal ability in growth media containing 50 μM Cd. Six strains out of 19 green algae and one out of five cyanobacteria removed more than 10% of total Cd from the medium. The marine green alga Chlorella sp. NKG16014 showed the highest removal of Cd 48.7% of total. Cd removal by NKG16014 was further quantitatively evaluated by measuring the amount of cell adsorption and intracellular accumulation. After 12 days incubation, 67% of the removed Cd was accumulated intracellularly and 25% of the Cd removed was adsorbed on the algal cell surface. The maximum Cd adsorption (q_max) was estimated to be 37.0 mg Cd (g dry cells)⁻¹ using the Langmuir sorption model. The Cd removal by freeze-dried NKG16014 cells was also determined. Cd was more quickly adsorbed by dried cells than that by living cells, with a q_max of 91.0 mg Cd (g dry cells)⁻¹.     

Factors controlling eicosapentaenoic acid production in semicontinuous cultures of marine microalgae

Three marine microalgal species with a high content of eicosapentaenoic acid (EPA), Phaeodactylum tricornutum, Isochrysis galbana and Porphyridium cruentum, were cultured semicontinuously in order to study the effect of renewal rate on EPA productivity. The percentage of EPA in total fatty acids increased with increasing renewal rates in nitrogen limited cultures, but while for Phaeodactylum tricornutum and Isochrysis galbana a plateau around 20–25% of total fatty acids was reached with renewal rates that were not nitrogen-limiting, in Porphyridium cruentum EPA percentage increased continuously with increasing renewal rate even for those cultures that were nitrogen sufficient. Maximal EPA productivities of4.6 mg L^-1 day^-1 for Isochrysis galbana and 5.2 mg L^-1 day^-1 for Phaeodactylum tricornutum were achieved with renewal rates of 20% and 30% respectively. On the other hand for Porphyridium cruentum maximal EPA productivity, 5.3 mg L^-1 day^-1, was obtained with the maximal renewal rate tested. Results indicate that different culture strategies should be adopted for the production of a particular polyunsaturated fatty acid depending on the microalgal species being used. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

Peptone,a low-cost growth-promoting nutrient for intensive animal cell culture

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×10⁷ cells ml^–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.     

Understanding and optimizing the addition of phytohormones in the culture of microalgae for lipid production

Some studies have described the use of phytohormones in microalgal culture for the production of biodiesel or selected fatty acids. However, no study has determined the amount of phytohormones that maximizes lipid yield. We determined the optimal concentration of auxins and gibberellins (which is between 40 and 60 μM) in two strains (Scenedemus abundans and Chlorella ellipsoidea) suitable for biodiesel production. More than 3‐fold increment was reached with S. abundans and near 7‐fold increment with C. ellipsoidea. Furthermore, this work suggests that the improved growth of the microalgae in the presence of the phytohormones was due to a reduction in the level of reactive oxygen species (ROS) in the cells. An economic analysis showed that, due to its low cost, auxin offers a positive cost‐benefit balance and therefore could be used at large scale. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1203–1211, 2016     

Evaluation of electro‐coagulation–flocculation for harvesting marine and freshwater microalgae

Although microalgae are considered as a promising feedstock for biofuels, the energy efficiency of the production process needs to be significantly improved. Due to their small size and low concentration in the culture medium, cost‐efficient harvesting of microalgae is a major challenge. In this study, the use of electro‐coagulation–flocculation (ECF) as a method for harvesting a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal species is evaluated. ECF was shown to be more efficient using an aluminum anode than using an iron anode. Furthermore, it could be concluded that the efficiency of the ECF process can be substantially improved by reducing the initial pH and by increasing the turbulence in the microalgal suspension. Although higher current densities resulted in a more rapid flocculation of the microalgal suspension, power consumption, expressed per kg of microalgae harvested, and release of aluminum were lower when a lower current density was used. The aluminum content of the harvested microalgal biomass was less than 1% while the aluminum concentration in the process water was below 2 mg L⁻¹. Under optimal conditions, power consumption of the ECF process was around 2 kWh kg⁻¹ of microalgal biomass harvested for Chlorella vulgaris and ca. 0.3 kWh kg⁻¹ for Phaeodactylum tricornutum. Compared to centrifugation, ECF is thus more energy efficient. Because of the lower power consumption of ECF in seawater, ECF is a particularly attractive method for harvesting marine microalgae. Biotechnol. Bioeng. 2011;108: 2320–2329. © 2011 Wiley Periodicals, Inc.     

Screening of marine bacteria for fucoidanases

Twenty-five strains of epiphytic marine bacteria isolated from the brown algaeFucus evanescens andChorda filum and fifty-three bacteria isolated from the sea cucumberApostichopus japonicus were screened for fucoidanases using fucoidans prepared from the brown algaeF. evanescens, Laminaria cichorioides, andL japonica. Eighteen bacterial epiphytes and thirty-eight bacterial isolates from the sea cucumber were found to contain fucoidanases, which were able to hydrolyze either all of the fucoidans studied or some of them. Bacteria of the generaCytophaga andAlteromonas/Pseudoalteromonas exhibited the highest fucoidanase activities, which, however, did not exceed the activity of fucoidanases from the already known sources.     

Microalgae for use in tropical aquaculture II: Effect of salinity on growth,gross chemical composition and fatty acid composition of three species of marine microalgae

The influence of salinity on the growth, gross chemical composition and fatty acid composition of three species of marine microalgae,Isochrysis sp.,Nannochloropsis oculata andNitzschia (frustulum), was investigated. There was no significant change in growth rate ofIsochrysis sp. andN. (frustulum) over the experimental range of salinity (10–35 ppt), whileN. oculata had a significantly slower growth rate only at 35 ppt. The ash content of all three species increased with increasing salinity. Two species,Isochrysis sp. andN. oculata, showed significant linear increases in total lipid content with increasing salinity over the range 10 to 35 ppt.N. (frustulum) showed significant linear decrease in total lipids, with the highest percentage at low salinity within the range 10–15 ppt. Variation in salinity had only a slight effect on the total protein, the soluble carbohydrate and chlorophylla content of all species. All species responded to change in salinity by modifying their cellular fatty acid compositions. Significant positive correlations were observed between increase in salinity and increase in the percentage ofcis-9-hexadecenoic acid [16:1 (n-7)] over the entire experimental range inN. (frustulum) and between 25–35 ppt inN. oculata. There were curved relationships between salinity and percentage of hexadecanoic acid [16:0] inN. oculata andN. (frustulum), with maxima within the range 25–30 ppt for both species. A curved relationship was found between salinity and percentage of eicosapentaenoic acid [20–5(n-3)], forN. (frustulum), with lowest percentages of the fatty acid within the range 25–30 ppt. There was no consistent pattern in the percentages of other major fatty acids as functions of salinity. The Northern Territory isolateN. (frustulum) was unusual in having a substantial increase in total fatty acids with decreasing salinity (85 mg g^–1 dry wt at 10 ppt compared with 33 mg g^–1 at 35 ppt). The optimum salinities for the production of maximum amount of lipids and the essential fatty acids 20:5(n-3) and/or 22:6(n-3) were as follows:25 ppt forIsochrysis sp. [22:6(n-3)]; 20–30 ppt forN. oculata [20:5(n-3)]; 10–15 ppt forN. (frustulum) [20:5(n-3) and 22:6(n-3)].Author for correspondence     

Growth physiology of a marine nitrogen-fixing cyanobacterium (Nodularia harveyana) in outdoor culture

The performance ofNodularia harveyana, a N₂-fixing cyanobacterium isolated from seawater, has been studied outdoors in two different culture systems: open pond (OP) and tubular photobioreactor (TPR). The productivity in both devices was influenced by areal density. The maximum yield obtained was 12.0 g (d.wt) m^–2 day^–1 in OP and 14.0 g (d.wt) m^–2 day^–1 in TPR in August, corresponding to the highest solar radiation received. In a month-long experiment with the cyanobacterium cultivated in TPR at high circulation speed, a net increase in productivity was obtained over that at low circulation speed. The influence of temperature on the productivity of the cultures grown in open ponds and tubular photobioreactors has been investigated. The higher productivity obtained in TPR compared to OP was attributed to its better controlled temperature conditions. In outdoor culture the maximum nitrogenase activity did not coincide with the maximum light intensity, but occurred in early afternoon. The amount of carbohydrate accumulated during the day probably influenced the rate of dark nitrogenase activity and its duration in the night.     

Semi-mass culture of the dinoflagellate Gymnodinium splendens as a live food source for the initial feeding of marine finfish larvae

A technique was developed for the semi-mass culture ofthe unarmored dinoflagellate, Gymnodiniumsplendens under laboratory conditions. A maximumcell density of 4600 to 6800 cells ml^–1 was observedwithin 8 to 11 days of culture. An initial feedingtest for 8 days with three important marine finfishlarvae showed that red spotted grouper, Epinephelus akaara preferred G. splendensfed 200 cells ml^–1 with 44% survival. The Japanesestripe knife jaw, Oplegnathus fasciatus,attained 22% survival fed a combination of G. splendensand rotifers (200 cells ml^–1 and 5ind. ml^–1, respectively). Red sea bream, Pagrusmajor larvae did not respond well to the initialfeeding of G. splendens alone. Red seabream were observed to be solely dependent on rotifers(5 ind. ml^–1) as initial food. Gymnodiniumsplendens may be used as a live food in the initialfeeding of red spotted grouper larvae (E. akaara) toreduce mortality and to further enhancegrowth during the critical first few days ofrearing.