Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> (Bohlin)

Microalgae cultures are receiving attention because of increasing biotechnological and biomedical production of active biomolecules. We evaluated various fertilizer-based culture media to scale up production of the marine microalga Phaeodactylum tricornutum for production of exocellular polysaccharides (EPS), soluble proteins, and cellular superoxide dismutase (SOD). The standard source of sodium nitrate was the same as that used in the synthetic f/2 culture medium and ammonium nitrate, urea, ammonium sulfate, and calcium nitrate as alternative sources of nitrogen. The maximum production of EPS was achieved in microalgae cells grown in the culture media containing 63 and 23% nitrogen from ammonium sulfate, and also in microalgae cells grown in the culture media containing 3% nitrogen from ammonium nitrate. The maximum production of cellular SOD was achieved in microalgae cells grown in the culture media containing 35 and 26% nitrogen from ammonium sulfate, and in the culture media containing 17% nitrogen from urea. The results suggest that it is possible to use a source of nitrogen, other than sodium nitrate, to scale up growth of P. tricornutum for production of EPS and SOD at reduced costs.     

A photobioreactor system for computer controlled cultivation of microalgae

A bioreactor system was developed for the cultivation of the microalgae Synechocystis sp. PCC6803 under controlled physiological conditions. The determination of the actual physiological state of the microalgae was provided by inline recording of chlorophyll fluorescence parameters. A feed-back loop was employed to keep the microalgae in a defined physiological state. For the construction of this feed-back loop, the temporal behaviour of the system was investigated using changes in light conditions (as caused by modulated UVB radiation) as input signal and chlorophyll fluorescence as output signal. The reproducibility of the responses was high. Kinetic analysis based on curve fitting revealed two time constants in the UVB-induced responses. The knowledge of these time constants was utilised for the development of an efficient feed-back loop which allows the cultivation of the microalgae in a defined physiological state. This new process strategy (called physiostat) was successfully tested. The performance in a culture of growing microalgae is shown.     

Screening of the culture media with different concentrations of nutrients for cultivation of the microalgae associated with the invertebrates of the White Sea

The growth and biomass accumulation of three microalgal strains of Desmodesmus (Scenedesmaceae, Chlorophyceae), 1Рm66В, 2Cl66E, and 3Dp86Е-1, isolated from the White Sea benthic invertebrates were studied under conditions of batch culture in different standard media (BG-11, Prat, Goldberg, Gromov, Tamiya, artificial seawater) and modified media. The culture condition, biomass accumulation, and uptake of nitrate and phosphate were recorded. A significant alkalization of the culture medium up to pH 10 has been observed during a vigorous growth of the microalgae. The most significant biomass accumulation has been recorded in BG-11 (in complete or modified medium with addition of artificial seawater), Tamiya, and Prat media. Addition of seawater did not affect the growth of Desmodesmus sp. in the nitrate-containing media, although that maintained growth of the microalgae in the nitrogen-lacking media without cell aggregation. The BG-11 medium appears suitable for isolation and cultivation of both symbiotic and free-living microalgae by all the tested features. The Prat medium is the best for maintaining the microalgal strains in living collection.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari's medium: 3.7, 37.0, 74.0, and 185.0mmol·L-1) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1stexperimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and [3-carotene contents in cells. In experimental series conducted respectively in Novem-ber 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

Microplates as a microreactor platform for microalgae research

High‐throughput platforms for microalgae screening are not yet commercially available. In this study, the feasibility of 96‐well microplates was analyzed for microalgae research. Equivalence among wells, as culture microreactors, was investigated in controlled high CO₂ conditions. Specific growth rates of two microalgae species, Scenedesmus sp. UTEX1589 and an environmental isolate, were significantly higher in border wells than in internal positions. Furthermore, growth rate gradients analyzed as contours throughout the platform were observed for Scenedesmus sp. However, the output variable exhibited high precision associated with a low coefficient of variation (CV), between 6.8 and 7.8%. In a demonstrative experiment to determine the effect of culture media dilution on six microalgae species, treatments were randomized in the central subset of a microplate. Results were consistent and statistically sound (CV 9.4–12.9%), and showed that microalgae species could grow with no detrimental effect in 50% (v/v) dilution of the culture medium. Provided border wells exclusion and a randomized design, 96‐well microplates are a practical and statistical robust platform for microalgae research. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:638–644, 2013     

微藻生物柴油技术的研究现状及展望

微藻生物柴油是一种优良的可再生新能源,对于解决人类面临的能源短缺和全球变暖两大危机具有潜在的重大战略意义。综述了微藻生物柴油的技术流程、油脂含量较高的微藻藻种、微藻生物柴油的最大技术瓶颈、提高微藻油脂总产量的方法、微藻的大规模培养、微藻的采收和微藻生物柴油的制取等方面的研究现状,并对微藻生物柴油未来的核心研究方向提出了初步见解。     

The potential of microalgal biotechnology: a review of production and uses of microalgae

An overview of the various aspects, promises and limitations of microalgal biotechnology is presented. The factors of importance in microalgal cultivation as well as the culture systems are briefly described. Microalgal biomasses can fulfil the nutritional requirements of aquatic larvae and organisms. The biochemical composition of algae can be improved by the manipulation of culture conditions. The nutritive value of the microalgal biomasses for human and animal consumption is also commented upon as well as some socio-economical aspects. Among the sources of required nutrients (N, P), wastewaters and manures can upgraded as culture media for microalgae the safety of which has to be evaluated. Harvesting of the biomass is one of the bottlenecks. The various techniques, physical, physico-chemical and biological are outlined and their feasibility and economic interest examined. Microalgal biomasses can be submitted to various technological transformations. Various processes are reviewed in the light of their effects on safety and nutritional value. The possible extraction of fine chemicals and the preparation of protein concentrates is also reported on. The various uses of microalgae lead to a possible competition, to be evaluated, between systems for the production of food, energy and chemicals. The review finally covers the application of genetic manipulation to microalgae.     

Microalgae immobilization: current techniques and uses

Information about advances in immobilization techniques and biotechnology use of freshwater and marine microalgae is scattered. This work aims to bring together the main recent research about the topic. Passive and active immobilization techniques used on microalgae are listed and described in the text. Effect of immobilization on growth and metabolism of the cells is also reviewed. Current uses of immobilized microalgae include metabolite production, culture collection handling, obtaining of energy and removing of undesired or valuable substances from media (nutrients, metals and different pollutant agents). Applications of immobilized microalgae in environmental aquatic research have been recently increased: novel immobilization techniques as well as the use of living microalgae as biosensors in electronic devices designed to measure toxicity of substances and effluents demonstrated to be a very promising topic in biotechnology research. Recent research pointed out the advantages of mixed bacterial-algal co-immobilized systems in water treatment plants. Application of immobilized systems to the production of non-contaminant energy (as H(2) obtained from algal cultures) is also an important topic to be explored in the next years.     

Effects of media NaCl concentration and osmolarity on the culture of early-stage porcine embryos and the viability of embryos cultured in a selected superior medium

One- and 2-cell porcine embryos were obtained from oviductal flushes and cultured for 96 hours in media with varied osmolarity that resulted from alterations in NaCl and sorbitol content. The viability of experimental embryos cultured to advanced stages was determined by comparison with that of the controls, noncultured embryos transferred to recipient gilts. The data suggest that variation in embryonic development in the experimental media is related to the NaCl concentration rather than to osmolarity. Increased NaCl concentration impairs development of the embryos to the advanced morula/blastocyst stages (P<0.001). There was no difference in the pregnancy rate between the recipients of cultured (45%) and noncultured (57%) embryos on Day 25. There was, however, a higher embryonic survival rate (P<0.05) within the control gilts.     

Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation

Microalgae are regarded as a potential biomass source for biofuel purposes. With regard to bioethanol production, microalgae seem to overcome traditional substrate drawbacks. Enzymatic activities are responsible for carbon allocation and hence for carbohydrate profiles. Enzyme activities may be manipulated by metabolic engineering; however, this goal may also be achieved by controlling environmental conditions of the culture system. We outline the key-enzymes as well as the main operational conditions applied to microalgae growth (inorganic nutrient supplementation, irradiance and temperature) that affect carbohydrate synthesis on microalgae and cyanobacteria. Normally, harsh conditions are needed for such a goal and thus, arrested microalgae growth may occur. Potential strategies to avoid arrested growth, while enhancing carbohydrate accumulation, were also pointed out in this review.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Season variability of iron effects on periodic culture of microalgae Dunaliella viridis Teod.

Effects of different iron concentrations (final concentrations of iron in Artari’s medium: 3.7, 37.0, 74.0, and 185.0 mmol·L⁻¹) on growth rate and contents of protein, triacylglycerides, and β-carotene in Dunaliella viridis cells at cultivation in different months were investigated. It was shown that the dose-dependent effects of iron were notable for season variability. In the 1^st experimental series (October, 2007), iron at researched concentrations did not affect growth rate of culture and protein, triacylglyceride, and β-carotene contents in cells. In experimental series conducted respectively in November 2007, December 2007, and February 2008, the dose-dependent stimulation of microalgae growth was observed. For each of these experimental series, there were particular dose dependences of protein, triacylglyceride, and β-carotene contents in microalgae cells at cultivation on media with iron at different concentrations. Meanwhile, for all of the four experimental series conducted in different months, variability of growth rate and analyzed parameters of microalgae Dunaliella viridis as control (cultivation without iron) was shown. It is suggested that these functional differences of control cultures of microalgae in different months caused variability in the dose-dependent effects of iron in a Dunaliella viridis culture. The possibility of iron usage for increasing microalgae biomass and for enriching it by β-carotene in Dunaliella viridis culture with initial low productivity and low β-carotene content is considered.     

New horizons in culture and valorization of red microalgae

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.     

Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

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.     

Open systems for the mass production of photoautotrophic microalgae outdoors: physiological principles

The major physiological principles involved in mass production of photoautotrophic microalgae outdoors relate to sustained trapping of solar energy in as high an efficiency as possible throughout the year.The tactics that should be employed for this goal include the improvement of suitable species, as well as developing culturing devices and proper management protocol aimed to facilitate efficient exploitation of the supper saturating photon flux densities existing outdoors.The most common system used today in industry for outdoor production of microalgae is the open raceway, in which stirring is provided by a paddle wheel. This mode of production suffers usually from many weaknesses, since it does not permit a satisfactory response to the two major variables that limit productivity outdoors — i.e.- solar irradiance and ambient temperature. Sustained production of algal mass the year round requires constant monitoring of the state of the culture and adjusting imputs accordingly. The readily controllable variables relate to mineral nutrients and carbon balance as well as to turbulent streaming in the culture and to the population density.The drawbacks of the open system relate in essence to the lack of temperature control and the long light-path which dictates maintenance of disadvantageously low cell concentrations. The open raceway thus falls short of the requirements necessary to insure sustained, year round high productivity outdoors.It is thus proposed that in the future, closed reactors may become the major production mode of microalgae outdoors.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

Screening of microalgal culture media for the presence of algicidal compounds and isolation and identification of two bioactive metabolites,excreted by the cyanobacteria <Emphasis Type="Italic">Nostoc insulare</Emphasis> and <Emphasis Type="Italic">Nodularia harveyana</Emphasis>

Culture medium extracts obtained from 115 culture media of 35 different microalgae species were screened for the presence of algicidal compounds, in particular for compounds which are cytotoxic to Arthrospira (Spirulina) laxissima. In agar plate diffusion tests and in a test system combining thin layer chromatography (TLC) with the use of an aqueous suspension of living A. laxissima cells as spray reagent, 14 microalgae species were found with cytotoxic activity of different intensity to A. laxissima. In a so-called TLC plate diffusion test, using A. laxissima and other microalgae as test organisms, the culture medium extracts of Nodularia harveyana and Nostoc insulare possessed the highest strength and range of algicidal activity. The algicidal compound in the culture medium extracts of Nodularia harveyana was shown to be norharmane (9H-pyrido(3,4-b)indole), a known indole alkaloid. The main algicidal compound in culture medium extracts of Nostoc insulare was identified as 4,4′-dihydroxybiphenyl. The possible applicability of both compounds as therapeutics or as useful agents for removing cyanobacterial water blooms or for developing new antifouling systems is discussed.     

Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h⁻¹ – Relative Fluorescence Unit h⁻¹), in comparison to batch cultivation (174.0 RFU h⁻¹) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L⁻¹.     

Diatom artificial medium (DAM): a new artificial medium for the diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> and other marine microalgae

Artificial media are used in physiological studies of microalgae to maintain consistent conditions from one experiment to another and these media must be adapted to the needs of the organism studied. The artificial medium, in this case named diatom artificial medium (DAM), was designed to maintain long-term cultures of Haslea ostrearia and 19 other planktonic microalgae, and to allow physiological studies related to metal metabolism. The biomass and biochemical composition of H. ostrearia grown in the DAM and in a modified Provasoli medium were compared to assess the suitability of this new artificial medium for the culture of this diatom. The DAM provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The DAM was tested with 19 other microalgae in order to widen its potential use, and 18 of the 19 showed a good adaptation to this medium. The chemical speciation of metals (Cd, Cu, Pb, Zn) was assessed using a speciation mathematical model. The presence of EDTA resulted in the total complexation of the trace metals implying that they were present in a sole chemical species in the DAM.     

Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae

The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.