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Microalgae have a growing recognition of generating biomass and capturing carbon in the form of CO

Background Microalgae are emerging as promising sustainable sources for biofuels, biostimulants in agriculture, soil bioremediation, feed and human nutrients. Nonetheless, the molecular mechanisms underpinning microalgae physiology and the biosynthesis of compounds of biotechnological interest are largely uncharacterized. This hinders the development of microalgae full potential as cell-factories. The recent application of omics technologies into microalgae research aims at unraveling these systems. Nevertheless, the lack of specific tools for analysing omics raw data generated from microalgae to provide biological meaningful information are hampering the impact of these technologies. The purpose of ALGAEFUN with MARACAS consists in providing researchers in microalgae with an enabling tool that will allow them to exploit transcriptomic and cistromic high-throughput sequencing data. Results ALGAEFUN with MARACAS consists of two different tools. First, MARACAS (MicroAlgae RnA-seq and Chip-seq AnalysiS) implements a fully automatic computational pipeline receiving as input RNA-seq (RNA sequencing) or ChIP-seq (chromatin immunoprecipitation sequencing) raw data from microalgae studies. MARACAS generates sets of differentially expressed genes or lists of genomic loci for RNA-seq and ChIP-seq analysis respectively. Second, ALGAEFUN (microALGAE FUNctional enrichment tool) is a web-based application where gene sets generated from RNA-seq analysis as well as lists of genomic loci from ChIP-seq analysis can be used as input. On the one hand, it can be used to perform Gene Ontology and biological pathways enrichment analysis over gene sets. On the other hand, using the results of ChIP-seq data analysis, it identifies a set of potential target genes and analyses the distribution of the loci over gene features. Graphical representation of the results as well as tables with gene annotations are generated and can be downloaded for further analysis. Conclusions ALGAEFUN with MARACAS provides an integrated environment for the microalgae research community that facilitates the process of obtaining relevant biological information from raw RNA-seq and ChIP-seq data. These applications are designed to assist researchers in the interpretation of gene lists and genomic loci based on functional enrichment analysis. ALGAEFUN with MARACAS is publicly available on https://greennetwork.us.es/AlgaeFUN/.

This study evaluates the comprehensive valorization of the byproducts derived from the two-phase olive oil elaboration process [i.e., olive washing water (OWW), olive oil washing water (OOWW), and olive mill solid waste (OMSW)] in a closed-loop process. Initially, the microalga

This study evaluates the comprehensive valorization of the byproducts derived from the two-phase olive oil elaboration process [i.e., olive washing water (OWW), olive oil washing water (OOWW), and olive mill solid waste (OMSW)] in a closed-loop process. Initially, the microalga Raphidocelis subcapitata was grown using a mixture of OWW and OOWW as the culture medium, allowing phosphate, nitrate, sugars, and soluble chemical oxygen demand removal. In a second step, the microalgal biomass grown in the mixture of washing waters was used as a co-substrate together with OMSW for an anaerobic co-digestion process. The anaerobic co-digestion of the combination of 75% OMSW-25% R. subcapitata enhanced the methane yield by 7.0 and 64.5% compared to the anaerobic digestion of the OMSW and R. subcapitata individually. This schedule of operation allowed for integration of all of the byproducts generated from the two-phase olive oil elaboration process in a full valorization system and the establishment of a circular economy concept for the olive oil industry., Barbara Rincón-Llorente thanks the “Ramon y Cajal ” Program (Contract RYC-2011-08783) from the Spanish Ministry of Economy and Competitiveness. The authors also thank the Regional Government of Andalucía, Junta de Andalucía, Consejeria de Economía Innovación, Ciencia y Empleo (Project of Excellence RNM-1970) for funding the research.

Astaxanthin is a valuable and highly demanded ketocarotenoid pigment, for which the chlorophycean microalga Haematococcus pluvialis is an outstanding natural source. Although information on astaxanthin accumulation in H. pluvialis has substantially advanced in recent years, its underlying molecular bases remain elusive. An integrative metabolic and transcriptomic analysis has been performed for vegetative Haematococcus cells, grown both under N sufficiency (green palmelloid cells) and under moderate N limitation, allowing concurrent active cell growth and astaxanthin synthesis (reddish palmelloid cells). Transcriptional activation was noticeable in reddish cells of key enzymes participating in glycolysis, pentose phosphate cycle and pyruvate metabolism, determining the adequate provision of glyceraldehyde 3 phosphate and pyruvate, precursors of carotenoids and fatty acids. Moreover, for the first time, transcriptional regulators potentially involved in controlling astaxanthin accumulation have been identified, a knowledge enabling optimization of commercial astaxanthin production by Haematococcus through systems metabolic engineering.

The likely use of continuous cultures of Chlorella vulgaris for flue gas abatement was studied. Firstly, no pH-controlled photo-bioreactors were operated in order to understand the tolerance of this microalgae to high CO2 concentrations in the airstream. Thus, the effect in biomass productivity, CO2 fixation and biochemical composition of different percentage of pure CO2 (ranging 1–12%) in the air supply was investigated. When the inlet CO2 concentration varied from 1 to 10%, no statistical differences were found (ANOVA, p, This work was financially supported by SOST-CO2 CENIT-project in collaboration with Inabensa S.A.

Microalgae production has gained attention in recent years as promising systems for CO2 abatement as well as a source of proteins, pigments, vitamins, lipids, and carbohydrates. Particularly, starch can be used for bioethanol production in a well-established fermentative process. The aim of this work was to maximize and model biomass productivity and CO2 assimilation in continuous cultures of Chlorella vulgaris. The following culture parameters were studied: dilution rate, pH, temperature, light intensity, and nitrogen supply. The proposed model (r2 = 0.95) predicted a maximum biomass productivity of 0.7 g L−1 d−1 and CO2 assimilation of 1.3 g L−1 d−1. The experimental data agreed with these predictions, resulting in a maximum biomass productivity of 0.67 g L−1 d−1 (resulting in a CO2 assimilation of 1.23 g L−1 d−1). In addition, the starch content was determined, and the results were used as input into a second model, which aimed at predicting starch accumulation during CO2 abatement processes (r2 = 0.84). This second model predicted a daily and continuous production of biomass with a maximum starch content of 0.25 g g−1 d−1 (25% dcw), but under different culture conditions than those found for maximizing biomass productivity and CO2 assimilation. The maximum starch content experimentally determined was 0.2 g g−1 d−1 (20% dcw). Thus, to implement a biological system for CO2 abatement coupled to starch accumulation, it is necessary to find a compromise between these two processes. Hence, although yield in both processes would be reduced, a simultaneous process for CO2 mitigation and starch production would be feasible., This research was supported by the scientific Project CENIT SOST-CO2–New sustainable industrial uses of CO2, with financing by INABENSA S.A.

A promising approach to CO2 abatement is the use of photosynthetic microorganisms. Different microalgae (Chloroccocum sp., Porphyridium purpureum, Scenedesmus vacuolatus) and cyanobacteria (Anabaena PCC7119, Anabaena PCC7120, Anabaena PCC 7937, Nostoc PCC 9202, Nostoc punctiforme) were cultivated in photobioreactors operated as chemostat, simulating light conditions analogous to those prevailing outdoors and compared on their ability to fix CO2 efficiently. Due to its high biomass productivity and CO2 fixation rate, Scenedesmus vacuolatus was selected and the effect of different culture parameters (i.e. dilution rate, temperature, pH and impinging irradiance on surface's photobioreactor) on its biomass productivity and CO2 fixation was further investigated. In optimal culture conditions, S. vacuolatus rendered 0.63 g biomass L− 1 d− 1, resulting 1.15 gCO2 assimilated L− 1 d− 1. Based on the data obtained in this study, a mathematical model was developed to describe growth and CO2 bio-fixation by S. vacuolatus. Finally, the potential of this microalga for carbon capture was further tested using synthetic flue gases as the source of CO2.

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers' demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on beta-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.

The nitrogen-fixing cyanobacterium Anabaena sp. ATCC 33047, which generates substantial amounts of exopolysaccharide, was immobilized by entrapment within the reticulate network of polyurethane foam discs. The immobilized Anabaena sp. system has been investigated as a potential biosorbent for the removal of cadmium from aqueous solutions. The results showed that it was a highly fast process, with 80 % of the adsorption taking place in the first 10 min, reaching full equilibrium in about 50 min. Data analysis indicated that the behaviour of the system accurately fits to a monolayer adsorption model (Langmuir isotherm). The maximal biosorption capacity determined for the immobilized Anabaena sp. system was as high as 162 mg Cd (II) per gram dry biomass. The outstanding properties established for immobilized Anabaena sp. in polyurethane foam underline the relevance of such a system as an alternative to current treatments of variety effluents or wastewater contaminated with cadmium.

A basic criterion in the selection of microalgae suitable as source of oil for biodiesel should be their actual capacity to produce lipids or, more properly, the fatty acid yield. Performance assessment of ten preselected microalgae under both batch and continuous culture points to the latter approach as the most adequate for evaluating fatty acid productivity. Differences were patent in continuous culture among strains that otherwise had analogous oil accumulation potential under batch culture. Some promising strains under batch culture (like Muriella aurantiaca and Monoraphidium braunii) exhibited, however, values for actual fatty acid productivity lower than 40 mg L-1d-1 in continuous regime. The analysis performed in photochemostat under continuous culture regime revealed the great potential of Chlorococcum olefaciens, Pseudokirchneriella subcapitata and Scenedesmus almeriensis as oil producing microalgae. Fatty acid productivity levels over 90 mg L-1d-1 were recorded for the latter strains under moderate nitrogen limitation, conditions which led to an enrichment in saturated and monounsaturated fatty acids, a more suitable profile as raw material for biodiesel. The continuous culture methodology employed represents a sound procedure for screening microalgae for biofuel production, providing a reliable evaluation of their fatty acid production capacity, under conditions close to those of outdoor production systems

The optimization of the operation, under the climatic conditions of southern Spain, of an experimental plant for β-carotene production by Dunaliella has been pursued. The effects of mixing, culture depth, cell density and dilution cycles on β-carotene and biomass productivity were studied under a semicontinuous culture regime in open tanks outdoors. Using 3 m2-surface containers, the highest productivity values, for both β-carotene and biomass, were recorded with a flow rate of 0.55 m s−1; 10 cm depth; 0.7 − 0.9 × 106cell ml−1, population density; and dilution cycles of two days. An average annual productivity of 1.65 g (dry wt) m−2 d−1 was estimated for Dunaliella biomass, being that for β-carotene of about 0.1 g m−2 d−1. Under these optimized conditions, experiments have been carried out at the Cadiz Bay with 20 m2-surface tanks during a whole-year cycle. The results obtained have validated this location and the operating conditions established as being most appropriate for efficient mass production of β-carotene rich D. salina.

The extent of biological CO2 fixation was evaluated for outdoor cultures of the cyanobacterium Anabaena sp. ATCC 33047. Culture conditions were optimized indoors in bubble-column photochemostats operating in continuous mode, subjected to irradiance cycles mimicking the light regime outdoors. Highest values achieved for CO2 fixation rate and biomass productivity were 1 and 0.6gL-1day-1, respectively. The comparison among different reactors operating simultaneously - open pond, horizontal tubular reactor and vertical flat-panel - allowed to assess their relative efficiency for the outdoor development of Anabaena cultures. Despite the higher volumetric CO2 fixation capacity (and biomass productivity) exhibited by the tubular photobioreactor, yield of the flat-panel reactor was 50% higher than that of the tubular option on a per area basis, reaching values over 35g CO2 fixed m-2d-1. The flat-panel reactor actually represents a most suitable system for CO2 capture coupled to the generation of valuable biomass by Anabaena cultures

Rotifers, used in aquaculture as feed for early larval stages, are poor in carotenoids compared with copepods, the natural most common prey item of marine fish larvae. A minimal sufficient level of carotenoids in fish larvae may be essential for a correct development. The freshwater microalga Muriellopsis sp. has been identified as a potential source of the carotenoid lutein with possible application in aquaculture. Lutein is a potent antioxidant, with potentially beneficial effects as a carotenoid for fish larvae. This study describes the lutein enrichment of the rotifer Brachionus sp. when incubated with homogeneously dispersed suspensions of freeze-dried, lutein-rich Muriellopsis sp. in seawater. A short enrichment time (2 or 2.5 h) resulted in a higher lutein content of the rotifers than a long enrichment time (13.5 or 24 h). Although the concentration of lutein in enriched rotifers, 0.06–0.16 mg (g dry weight) 1 was low compared with its content in Muriellopsis sp. [about 3 mg (g dry weight) 1 ], it was in the range reported for natural copepod populations. Therefore, we conclude that freeze-dried biomass of Muriellopsis sp. can be used to enrich rotifers with lutein, providing an adequate level of this carotenoid for marine fish larvae.

Cytoplasmic membranes prepared from nitrate-grown Anacystis nidulans cells exhibit a Mg2+-dependent protein kinase activity able to phosphorylate in vitro plasma membrane polypeptides with molecular masses of 98, 93, 83, 47, 44 and 31 kDa. The protein kinase activity was inhibited in cytoplasmic membrane preparations from nitrate-grown cells which had been exposed to ammonium for 5 min. Parallely, ammonium exposure also resulted in a more than two-fold activation of an alkaline phosphatase activity present in the soluble fraction. These results are discussed in relation to the well-known inhibition by ammonium of nitrate transport activity, and a hypothesis for the regulatory mechanism involved is presented.

The effect of operation regime and culture system on carotenoid productivity by the halotolerant alga Dunaliella salina has been analyzed. Operation strategies tested included batch and semi continuous regime, as well as a two-stage approach run simultaneously in both, open tanks and closed reactor. The best results were obtained with the closed tubular photobioreactor. The highest carotenoid production (328.8mgcarotenoidl-1 culture per month) was achieved with this culture system operated following the two-stage strategy. Also, closed tubular photobioreactor provided the highest carotenoid contents (10% of dry weight) in Dunaliella biomass and β-carotene abundance (90% of total carotenoids) as well as the highest 9-cis to all-trans β-carotene isomer ratio (1.5 at sunrise). © 2010 Elsevier B.V., The skilful technical assistance of Dr. R. Rodriguez is very much appreciated. This work was supported by Comisión Interministerial de Ciencia y Tecnología (grant no. IFD 1997-1780 co-financed with FEDER funds from EU), IFAPA (CO3-125) and Plan Andaluz de Investigación (group no. CVI131), Spain.

The influence of nitrate and ammonium assimilation on glycogen metabolism has been determined in intact Anacystis nidulans cell actively fixing CO2. Assimilation of nitrate or ammonium resulted in significant decreases in both the incorporation into glycogen of newly fixed carbon and the accumulation of glycogen by the cells, the magnitude of these effects depending on the light intensity. The depression in glycogen synthesis induced by nitrogen assimilation was more marked at low light intensity, and especially when ammonium was the nitrogen source. Under these conditions, specific radioactivity of the glycogen pool was particularly high, indicating enhanced turnover of glycogen. Thus, in addition to a more general depressing effect of nitrogen assimilation on the carbon flow to glycogen, degradation of glycogen appears to be stimulated by ammonium assimilation at low (but not at high) light intensity.

Summary The effect of free Cd 2+ ions on growth, photosynthesis, photosynthetic pigments (chlorophyll and phycobiliproteins), nitrate and ammonium uptake and divalent cation (Mg 2+ , Ca 2+ ) content in Anabaena PCC 7119 grown in a medium containing nitrate or ammonium was investigated. After 96 h of exposure, the metal causes an inhibition of photosynthesis in cells of Anabaena PCC 7119 growing in NH 4 + and NO 3 - media and a decrease in the photosynthetic pigments. Likewise, Cd 2+ toxicity elicited a reduction in the intracellular concentrations of Mg 2+ and Ca 2+ and an inhibition in ammonium uptake, resulting in changes in the cellular structure. The possible relation between the differential response in each culture medium and pH is discussed.

Nitrogenase activity of Anabaena PCC 7119 is inhibited under conditions of boron deficiency. To elucidate the mechanisms of this inhibition, this study examined how the deficiency of boron affected photosynthesis, photosynthetic pigments, the enzymes of the oxidative pentose phosphate pathway, and respiration of Anabaena PCC 7119 cultures. After 24 to 48 hours of boron deficiency, reductions in photosynthetic O(2) evolution and in CO(2) fixation were observed. At the same time, the activities of oxidative pentose phosphate pathway enzymes and respiration increased significantly with boron deficiency. No change was observed in these processes when assays were performed after 4 to 6 hours of deficiency, a time at which nitrogenase activity was severely inhibited. These results suggest that the requirement for boron in N(2) fixation is independent of its effects on photosynthesis and reductant supply.

Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2g (dry weight) m(-2) d(-1) or 80 gm(-3) d(-1)) was achieved at 38 cm s(-1), flow rate; 2 x 10(9) cells l(-1), initial population density; 25 degrees C, temperature; semi-continuous regime, keeping a cell density interval between 2 x 10(9) and over 4 x 10(9) cells l(-1). Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular beta-carotene level increased significantly during the light period, as also did that of lutein. The rise in the beta-carotene level could be accounted by the 9-cis-isomer, with all-trans-beta-carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of beta-carotene amounted to 1.5, with over 60% of total beta-carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of beta-carotene and lutein.

The effect of boron on heterocystous and nonheterocystous dinitrogen fixing Cyanobacteria was examined. The absence of boron in culture media inhibited growth and nitrogenase activity in Nodularia sp., Chlorogloeopsis sp., and Nostoc sp. cultures. Examinations of boron-deficient cultures showed changes in heterocyst morphology. However, cultures of nonheterocystous Cyanobacteria, Gloeothece sp. and Plectonema sp., grown in the absence of boron did not show any alteration in growth or nitrogenase activity. These results suggest a requirement of boron only by heterocystous Cyanobacteria. A possible role for this element in the early evolution of photosynthetic organisms is proposed.

Summary The requirement of sodium for photosynthesis and nitrate assimilation in a mutant of Nostoc muscorum, unable to fix dinitrogen, was examined. After 24 h of culture, sodium deficient cells showed changes in the contents of photosynthetic pigments as well as in photosynthesis and nitrate assimilation. Both CO2 consumption and photosynthetic oxygen evolution were reduced when cells were cultured for 24 h in absence of sodium. Similarly nitrate uptake was inhibited in sodium deficient cells. Nitrate reductase activity however was increased by 37% under these conditions. When assays were performed after 2 h of deficiency only the photosynthetic activities (both photofixation of CO2 and photoevolution of O2) were altered by the absence of sodium. These results suggest that the impairment of nitrate assimilation is a secondary effect of sodium deficiency and that the requirement of sodium for photosynthesis is independent of its effects on nitrate assimilation.

The effect of boron on nitrogenase activity has been studied. When cells were dependent on N(2) fixation, the lack of boron inhibited nitrogenase activity. However, under anaerobic conditions or in the presence of Na-dithionite this effect was not observed. Nitrogenase synthesis was not affected by boron deficiency. Similarly, the heterocyst number was not altered. Examination of boron-deficient cultures showed, however, some dramatic changes in heterocyst morphology. The increased activity of those enzymes related to the maintaining of the low intracellular level of toxic oxygen species (superoxide dismutase, catalase, and peroxidase) support our hypothesis of the role of boron in heterocyst envelope stabilization.

We describe a simple and efficient non-chromatographic method for the purification of murine IgG3 and IgM monoclonal antibodies (MAbs) which takes advantage of their euglobulin properties. Following filtration, ascitic fluid is dialysed against demineralized water and centrifuged at 22,000 X g for 30 min. The resulting precipitate is dissolved in a high salt buffer (0.1 M Tris-HCl, 1 M NaCl, pH 8). A second cycle of dialysis and centrifugation yields a product of high purity. Nine IgG3 MAbs and eight IgM MAbs were purified by this procedure. Recovery was greater than 90% for seven of nine IgG3 MAbs. It was less reproducible for IgM MAbs and ranged from 40% to greater than 90% depending on antibody and batch. Purity was assessed by SDS-polyacrylamide gel electrophoresis. The purified immunoglobulin was essentially free of albumin, transferrin, and other mouse ascites proteins. No loss of antibody function was observed.