Ana Otero

Two-stage cultures for the production of Astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis.

Abstract The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 105 cells ml−1, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l−1) KNO3 0.41, Na2HPO4 0.03, MgSO4 · 7H2O 0.246, CaCl2 · 2H2O 0.11, (in mg l−1) Fe(III)citrate · H2O 2.62, CoCl2 · 6H2O 0.011, CuSO4 · 5H2O 0.012, Cr2O3 0.075, MnCl2 · 4H2O 0.98, Na2MoO4 · 2H2O 0.12, SeO2 0.005 and (in μg l−1]) biotin 25, thiamine 17.5 and B12 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis.

Quorum quenching activity in Anabaena sp. PCC 7120: identification of AiiC, a novel AHL-acylase

Many bacteria use quorum sensing (QS) to coordinate responses to environmental changes. In Gram-negative bacteria, the most extensively studied QS systems rely on the use of N-acylhomoserine lactones (AHLs) signal molecules. Some bacteria produce enzymes that are able to inactivate AHL signals produced by other bacteria and hence interfere with QS-mediated processes via a phenomenon known as quorum quenching. Acylase-type AHL degradation activity has been found in the biomass of the filamentous nitrogen-fixing cyanobacterium Anabaena (Nostoc) sp. PCC 7120, being absent from the culture media. The gene all3924 has been identified and cloned whose product exhibits homology to the acylase QuiP of Pseudomonas aeruginosa PAO1, demonstrating that it is at least partially responsible for the AHL-acylase activity. The recombinant enzyme, which was named auto-inducer inhibitor from Cyanobacteria (AiiC), shows broad acyl-chain length specificity. Because the presence of AHLs in the biomass of nitrogen-fixing cultures of Anabaena sp. PCC 7120 has been described recently, AiiC could represent a self-modulatory system to control the response to its own QS signals but could also be involved in the interference of signalling within complex microbial communities in which Cyanobacteria are present.

Acylhomoserine lactone production and degradation by the fish pathogen Tenacibaculum maritimum, a member of the Cytophaga–Flavobacterium–Bacteroides (CFB) group

Tenacibaculum maritimum (formerly Flexibacter maritimus) is a filamentous, biofilm-forming member of the Cytophaga-Flavobacterium-Bacteroides group (or Bacteroidetes), which causes the widely distributed marine fish disease tenacibaculosis. A search for N-acylhomoserine lactones (AHLs) quorum-sensing (QS) signals in the culture media of nine representative strains of this species using different biosensor strains revealed the presence of short-type AHL activity in all of them. N-butyryl-L-homoserine lactone (C4-HSL) was identified in T. maritimum NCIMB2154(T) by LC-MS. A degradation activity for long-acyl AHLs (C10-HSL) was subsequently demonstrated in T. maritimum NCIMB2154(T). The acidification of the culture medium after degradation did not allow the recovery of C10-HSL, which indicates a possible acylase-type degradation activity. Even though the physiological processes under the control of AHL-mediated QS in T. maritimum need to be further characterized, this discovery extends the paradigm of AHL-mediated QS signalling beyond the Proteobacteria and reinforces its ecological significance.

Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis

Haematococcus pluvialis was cultured under N– and Mg+2-deficient conditions with two light intensities: 40 and 230 μmol m2 s−1. Highest astaxanthin concentration, 49.5 μg·ml−1, was obtained when high light was applied under N-deficient conditions. N-deficiency has a greater effect than high light intensity on astaxanthin synthesis by exerting a stronger blocking effect on cell division. The effect of high light was synergetic with the other stress conditions in stimulating the synthesis of astaxanthin. Mg+2 deficiency also stimulated the synthesis of astaxanthin but produced lower concentrations: 7 and 26 μg·ml−1 for low and high light intensities respectively. When both N and Mg+2 were absent from the culture media the concentration of astaxanthin was lower than with N-deficiency alone but higher than with Mg+2-deficiency.

Interactions between irradiance and nutrient availability during astaxanthin accumulation and degradation in Haematococcus pluvialis

Fully synchronised germination of Haematococcus pluvialis astaxanthin-replete aplanospores was induced by transfer to nitrogen-sufficient conditions under either high or low light intensities, and growth, pigment content and nitrogen consumption were monitored during the cell cycle. No germination of the aplanospores was achieved in the absence of nitrate, even when cells were transferred at low light intensities. On the other hand, cell density and chlorophyll concentration increased dramatically and astaxanthin concentration decreased in N-sufficient cultures due to the germination of 100% of the aplanospores, as demonstrated by flow cytometry. No significant effect of light intensity was observed on the degradation of astaxanthin during germination. In germinated cultures, nitrogen was depleted more rapidly under high light conditions, which resulted in earlier entry into the aplanospore stage and accumulation of astaxanthin. Germination of aplanospores accompanied by astaxanthin degradation could also be obtained in the dark in nutrient-sufficient conditions although at a much lower efficiency. The results demonstrate that nutrient availability is the main factor controlling the transition between red and green stages of H. pluvialis, with astaxanthin being accumulated only when cell division has ceased. High light levels accelerate the process by increasing the rate of nutrient depletion and providing more energy for astaxanthin synthesis.

Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light:dark cycle

Nannochloropsis gaditana was grown in semicontinuous culture with a circadian light:dark cycle in a flat-panel photobioreactor. The microalga had a maximal protein content (3 pg cell−1) after 6 h light and then only storage compounds were accumulated that were consumed during the dark phase. Carbohydrates reached their maximum value after 8 h (0.8 pg cell−1) and lipids after 12 h light (2.5 pg cell−1). The results demonstrated that young or adult microalgae might be obtained according to the time of day.

Changes in the nutrient composition of Tetraselmis suecica cultured semicontinuously with different nutrient concentrations and renewal rates

Abstract The marine microalga Tetraselmis suecica was cultured semicontinuously with two nutrient concentrations: 2 and 4 mmol N l −1 and five rates of daily renewal of the culture media in the range 10–50%, in order to study the changes in the nutrient composition of the cells produced under such conditions. An increase in the renewal rate produced an increase in protein and lipid content of the biomass to the detriment of carbohydrate content, the only fraction accumulated by T. suecica as energetic reserve under nitrogen deficient conditions. Cellular content of these three fractions did not accurately reflect the relative composition, since total organic content of the cells decreased with increasing renewal rates attenuating the increase in protein and lipid contents. As a result, cell caloric content remained almost constant. The degree of unsaturation of fatty acids increased with increasing renewal rate, with a two-fold increase of the percentage of total n − 3 fatty acids. On the other hand, the percentage of eicosapentaenoic acid (EPA) decreased with increasing renewal rate indicating a different control of the metabolism of this long-chain polyunsaturated fatty acid in T . suecica . Results demonstrate the efficiency of the use of the semicontinuous culture technique for the production of microalgal biomass of optimal biochemical composition being especially useful for the study of the nutritional requirements of filter-feeding marine organisms.

Patents on quorum quenching: interfering with bacterial communication as a strategy to fight infections.

Numerous bacterial functions, such as virulence and biofilm formation, are controlled by a cell densitydependent communication mechanism known as Quorum Sensing (QS), in which small diffusible molecules are released, allowing bacteria to coordinate their behavior once a minimal effective quorum has been reached. The interference with these signaling systems, also known as Quorum Quenching (QQ), represents a promising strategy to tackle bacterial infections. The growing interest in this approach is reflected by the increasing number of patents within the field (45 up to now), especially in the last few years, as shown by patent applications published since 2009. The fact that biofilm formation is also controlled by QS systems expands the application of QQ to clinically-relevant biofilms such as those responsible for periodontal disease. Moreover, since biofilms increase bacterial resistance to antimicrobials, QQ could represent a new way to fight some of the most recurrent human pathogens, such as nosocomial multiresistant strains, and this deserves further exploration, especially through more proofs of concept. In this article we review the best known QS and QQ systems to date and we describe recent patents on the interference with this type of bacterial communication.

Growth Rate of the microalga Tetraselmis suecica changes the biochemical composition of Artemia species.

Abstract: The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved.