Role of microalgae in conditioning water in penaeid larval culture

Microalgae play an important role in conditioning water quality for penaeid larval culture. Recently it has been demonstrated that a modification of the green water larval culture system (Ling, 1969) for Macrobrachium allows the production of post larvae without any water change, despite extensive use of artificial feeds (Ang and Cheah, 1986). Increase of toxic metabolites such as ammonia and nitride are also common in penaeid larval culture, especially where excessive amounts of artifial feeds are employed. Present work examines the use of six marine microalgae at four cell concentrations as a "biological filter" system, to control and detoxify levels of ammonia and nitrite in P. monodon larval culture water whilst using artificial diet. Preliminary results indicate that amongst the six algal species tested, C. japonica at 1000 cell μlˉ¹ was most effective in reducing accumulated toxic metabolites from an unchanged culture water environment.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

The vertical microdistribution of cyanobacteria and green algae within desert crusts and the development of the algal crusts

Substantial amounts of algal crusts were collected from five different desert experimental sites aged 42, 34, 17, 8 and 4 years, respectively, at Shapotou ( China) and analyzed at a 0.1 mm microscale of depth. It was found that the vertical distribution of cyanobacteria and microalgae in the crusts was distinctly laminated into an inorganic-layer (ca. 0.00 - 0.02 mm, with few algae), an algae-dense-layer ( ca. 0.02 - 1.0 mm) and an algae-sparse-layer ( ca. 1.0 - 5.0 mm). It was interesting to note that in all crusts Scytonema javanicum Born et Flah ( or Nostoc sp., cyanobacterium), Desmococcus olivaceus (Pers ex Ach., green alga) Laundon and Microcoleus vaginatus Gom. ( cyanobacterium) dominated at the depth of 0.02 - 0.05, 0.05 - 0.1 and 0.1 - 1.0 mm, respectively, from the surface. Phormidium tenue Gom. ( or Lyngbya cryptovaginatus Schk., cyanobacterium) and Navicula cryptocephala Kutz.( or Hantzschia amphioxys (Ehr.) Grun. and N. cryptocephala together, diatom) dominated at the depth of 1.0 - 3.0 and 3.5 - 4.0 mm, respectively, of the crusts from the 42 and 34 year old sites. It was apparent that in more developed crusts there were more green algae and the niches of Nostoc sp., Chlorella vulgaris Beij., M. vaginatus, N. cryptocephala and fungi were nearer to the surface. If lichens and mosses accounted for less than 41.5% of the crust surface, algal biovolume was bigger when the crust was older, but the opposite was true when the cryptogams other than algae covered more than 70%. In addition to detailed species composition and biovolume, analyses of soil physicochemical properties, micromorphologies and mineral components were also performed. It was found that the concentration of organic matter and nutrients, electric conductivity, silt, clay, secondary minerals were higher and there were more micro-beddings in the older crusts than the less developed ones. Possible mechanisms for the algal vertical microdistribtion at different stages and the impact of soil topography on crust development are discussed. It is concluded that biomethods ( such as fine species distribution and biovolume) were more precise than mineralogical approaches in judging algal crust development and thus could be a better means to measure the potentiality of algal crusts in desert amelioration.

The CO2 microalgae biorefinery: high value products and biofuels using halophilic microalgae in the “D-Factory”

Fuel-only algal systems are not economically feasible because yields are too low and costs too high for producing microalgal biomass compared to using agricultural residues e.g. straw. Biorefineries which integrate biomass conversion processes and equipment to produce fuels, power and chemicals from biomass, offer a solution. The CO2 microalgae biorefinery (D-Factory) is a 10 million Euro FP7-funded project which will cultivate the microalga Dunaliella in highly saline non-potable waters in photobioreactors and open raceways and apply biorefinery concepts and European innovations in biomass processing technologies to develop a basket of compounds from Dunaliella biomass, including the high value nutraceutical, β-carotene, and glycerol. Glycerol now finds markets both as a green chemical intermediate and as a biofuel in CHP applications as a result of novel combustion technology. Driving down costs by recovering the entire biomass of Dunaliella cells from saline cultivation water poses one of the many challenges for the D-Factory because Dunaliella cells are both motile, and do not possess an external cell wall, making them highly susceptible to cell rupture. Controlling expression of desired metabolic pathways to deliver the desired portfolio of compounds flexibly and sustainably to meet market demand is another. The first prototype D-Factory in Europe will be operational in 48 months, and will serve as a robust manifestation of the business case for global investment in algae biorefineries and in large-scale production of microalgae.

Microalgae taxonomy and breeding

Microalgae are a diverse group of organisms that form the basic component of many eco­- systems. The systemic classification of algae is primarily based on their pigment composition and is divided into nine classes. The largest groups are Chlorophyceae (green algae), Phaeophyceae (brown algae), Pyrrophyceae (dinoflagellates), Chrysophyceae (golden brown algae), Bacillariophyceae (diatoms) and Rhodo­ phyceae (red algae). It has been estimated that between 22,000 and 26,000 species exist (Norton et al., 1996), of which only a few species have been identified to be useful for commercial application, such as Spirulina, Chlorella, Haematococcus, Dunaliella, Botryococcus, Phaeodactylum and Porphyridium. Several other species that are also cultivated commercially for the hatcheries in the aquaculture field include Chaetoceros, Crypthecodinium, Isochrysis, Nannochloris, Nitzschia, Schizo­chytrium, Tetraselmis and Skeletonema (Raja et al., 2008).

Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plants

A discussion of the most interesting results obtained in our laboratories, during the supercritical CO(2) extraction of bioactive compounds from microalgae and volatile oils from aromatic plants, was carried out. Concerning the microalgae, the studies on Botryococcus braunii and Chlorella vulgaris were selected. Hydrocarbons from the first microalgae, which are mainly linear alkadienes (C(23)-C(31)) with an odd number of carbon atoms, were selectively extracted at 313 K increasing the pressure up to 30.0 MPa. These hydrocarbons are easily extracted at this pressure, since they are located outside the cellular walls. The extraction of carotenoids, mainly canthaxanthin and astaxanthin, from C. vulgaris is more difficult. The extraction yield of these components at 313 K and 35.0 MPa increased with the degree of crushing of the microalga, since they are not extracellular. On the other hand, for the extraction of volatile oils from aromatic plants, studies on Mentha pulegium and Satureja montana L were chosen. For the first aromatic plant, the composition of the volatile and essential oils was similar, the main components being the pulegone and menthone. However, this volatile oil contained small amounts of waxes, which content decreased with decreasing particle size of the plant matrix. For S. montana L it was also observed that both oils have a similar composition, the main components being carvacrol and thymol. The main difference is the relative amount of thymoquinone, which content can be 15 times higher in volatile oil. This oxygenated monoterpene has important biological activities. Moreover, experimental studies on anticholinesterase activity of supercritical extracts of S. montana were also carried out. The supercritical nonvolatile fraction, which presented the highest content of the protocatechuic, vanilic, chlorogenic and (+)-catechin acids, is the most promising inhibitor of the enzyme butyrylcholinesterase. In contrast, the Soxhlet acetone extract did not affect the activity of this enzyme at the concentrations tested. (C) 2011 Elsevier B.V. All rights reserved.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.

Sea bream (Sparus aurata) intensive larval rearing : effects of microalgae and phages addition on the performance of the larvae

Máster Oficial en Cultivos Marinos. Trabajo presentado como requisito parcial para la obtención del Título de Máster Oficial en Cultivos Marinos, otorgado por la Universidad de Las Palmas de Gran Canaria (ULPGC), el Instituto Canario de Ciencias Marinas (ICCM), y el Centro Internacional de Altos Estudios Agronómicos Mediterráneos de Zaragoza (CIHEAM)

Vertical microalgae fluxes at the mooring station LOMO-2 over the Lomonosov Ridge in the period from September 15, 1995 till August 16, 1996

The first studies of microalgae fluxes over the Lomonosov Ridge in the northern Laptev Sea were carried out with a sediment trap at the year-long mooring station LOMO-2, installed at 150 m depth from September 15, 1995 to August 16, 1996. These studies demonstrated essential seasonal variations of vertical microalgae flux. It was shown that in summer diverse flora (composed mainly of cryophylic diatoms) growed intensively beneath the permanent ice cover. Strongly pronounced seasonal variations of microalgae growth correlate closely with solar radiation. Exactly during the maximum insolation period, from the middle of July until the end of September, the microalgae flux was hundreds of times higher than that in the rest of the year. Summer values of the microalgae flux over the Lomonosov Ridge in the northern Laptev Sea were similar to those in the Weddell Sea (Antarctic) and exceeded summer flux values in the Norwegian and Greenland Seas and in the St. Anna Trough (northwestern Kara Sea).

Experimental nutrient enrichment causes complex changes in seagrass, microalgae, and macroalgae community structure in Florida Bay

We evaluated how changes in nutrient supply altered the composition of epiphytic and benthic microalgal communities in a Thalassia testudinum (turtle grass) bed in Florida Bay. We established study plots at four sites in the bay and added nitrogen (N) and phosphorus (P) to the sediments in a factorial design. After 18, 24, and 30 months of fertilization we measured the pigment concentrations in the epiphytic and benthic microalgal assemblages using high performance liquid chromatography. Overall, the epiphytic assemblage was P-limited in the eastern portion of the bay, but each phototrophic group displayed unique spatial and temporal responses to N and P addition. Epiphytic chlorophyll a, an indicator of total microalgal load, and epiphytic fucoxanthin, an indicator of diatoms, increased in response to P addition at one eastern bay site, decreased at another eastern bay site, and were not affected by P or N addition at two western bay sites. Epiphytic zeaxanthin, an indicator of the cyanobacteria/coralline red algae complex, and epiphytic chlorophyll b, an indicator of green algae, generally increased in response to P addition at both eastern bay sites but did not respond to P or N addition in the western bay. Benthic chlorophyll a, chlorophyll b, fucoxanthin, and zeaxanthin showed complex responses to N and P addition in the eastern bay, suggesting that the benthic assemblage is limited by both N and P. Benthic assemblages in the western bay were variable over time and displayed few responses to N or P addition. The contrasting nutrient limitation patterns between the epiphytic and benthic communities in the eastern bay suggest that altering nutrient input to the bay, as might occur during Everglades restoration, can shift microalgal community structure, which may subsequently alter food web support for upper trophic levels.

The effect of a reduction of nitrogen and phosphorus to eutrophic and mesotrophic conditions towards silver nanoparticle (AgNP) on freshwater green algae species

We investigated the sensitivity of algae towards silver nanoparticles with OECD test medium and lower nutrient concentrations under standard test conditions to improve comparability and to exclude any other confounding factor aside nutrient levels. Two unicellular freshwater microalgae Desmodesmus subspicatus and Raphidocelis subcapitata were chosen due to their status as standard test organisms for the algae growth inhibition test and the response to changes in nutrient supply was compared. The original medium was used as the reference (standard). For the other four media, the amount of either nitrogen or phosphorus in the medium was lowered from half (50%) to one-fourth (25 %) of that of the OECD guideline, resulting in the following media: 50% N, 25% N, 50% P and 25% P medium. As test substance, the OECD reference material NM-300K was used. For this reason, the characterization of AgNP was done using DLS and Absorption spectra (UV/vis). Actual silver concentrations and ionic silver concentrations were measured at the highest test concentration used (100 µg Ag L-1) in R. subcapitata treatments only to reduce the number of samples. All tests were run according to the OECD guideline 201 with sterilized 50 mL cell culture flask. Each medium was tested using the test conditions for culturing with 3 replicates. Test concentrations for both algae species were 0, 25, 50 and 100 µg Ag L-1 for OECD, 50% P and 25% P while for both N reductions, the silver concentrations were 0, 10, 25 and 100 µg Ag L-1. Samples for determining the algal density were taken at every 24 h.

Extraction, partial characterization and susceptibility to Hg2+ of acid phosphatase from the microalgae Pseudokirchneriella subcapitata.

Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 μM Hg2+ .

Immobilized Microalgae for Nutrient Recovery from Source Separated Urine

Shortages in supply of nutrients and freshwater for a growing human population are critical global issues. Traditional centralized sewage treatment can prevent eutrophication and provide sanitation, but is neither efficient nor sustainable in terms of water and resources. Source separation of household wastes, combined with decentralized resource recovery, presents a novel approach to solve these issues. Urine contains within 1 % of household waste water up to 80 % of the nitrogen (N) and 50 % of the phosphorus (P). Since microalgae are efficient at nutrient uptake, growing these organisms in urine might be a promising technology to concomitantly clean urine and produce valuable biomass containing the major plant nutrients. While state-of-the-art suspension systems for algal cultivation have mayor shortcomings in their application, immobilized cultivation on Porous Substrate Photobioreactors (PSBRs) might be a feasible alternative. The aim of this study was to develop a robust process for nutrient recovery from minimally diluted human urine using microalgae on PSBRs. The green alga Desmodesmus abundans strain CCAC 3496 was chosen for its good growth, after screening 96 algal strains derived from urine-specific isolations and culture collections. Treatment of urine, 1:1 diluted with tap water and without addition of nutrients, was performed at a light intensity of 600 μmol photons m-2 s-1 with 2.5 % CO2 and at pH 6.5. A growth rate of 7.2 g dry weight m-² day-1 and removal efficiencies for N and P of 13.1 % and 94.1 %, respectively, were determined. Pre-treatment of urine with activated carbon was found to eliminate possible detrimental effects of pharmaceuticals. These results provide a basis for further development of the technology at pilot-scale. If found to be safe in terms human and environmental health, the biomass produced from three persons could provide the P for annual production of 31 kg wheat grain and 16 kg soybean, covering the caloric demand in food for almost one month of the year for such a household. In combination with other technologies, PSBRs could thus be applied in a decentralized resource recovery system, contributing to locally close the link between sanitation and food production.