Microalgal Carotenoids: A Review of Production, Current Markets, Regulations, and Future Direction.

Microalgae produce a variety of compounds that are beneficial to human and animal health. Among these compounds are carotenoids, which are microalgal pigments with unique antioxidant and coloring properties. The objective of this review is to evaluate the potential of using microalgae as a commercial feedstock for carotenoid production. While microalgae can produce some of the highest concentrations of carotenoids (especially astaxanthin) in living organisms, there are challenges associated with the mass production of microalgae and downstream processing of carotenoids. This review discusses the synthesis of carotenoids within microalgae, their physiological role, large-scale cultivation of microalgae, up- and down-stream processing, commercial applications, natural versus synthetic carotenoids, and opportunities and challenges facing the carotenoid markets. We emphasize legal aspects and regulatory challenges associated with the commercial production of microalgae-based carotenoids for food/feed, nutraceutical and cosmetic industry in Europe, the USA, the People's Republic of China, and Japan. This review provides tools and a broad overview of the regulatory processes of carotenoid production from microalgae and other novel feedstocks.

Exploring the microbiome of the "star" freshwater diatom Asterionella formosa in a laboratory context.

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.

A novel unsaturated ß-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105.

Ulvans are cell wall matrix polysaccharides in green algae belonging to the genus Ulva. Enzymatic degradation of the polysaccharide by ulvan lyases leads to the production of oligosaccharides with an unsaturated ß-glucuronyl residue located at the non-reducing end. Exploration of the genomic environment around the Nonlabens ulvanivorans (previously Percicivirga ulvanivorans) ulvan lyase revealed a gene highly similar to known unsaturated uronyl hydrolases classified in the CAZy glycoside hydrolase family 105. The gene was cloned, the protein was overexpressed in Escherichia coli, and enzymology experiments demonstrated its unsaturated ß-glucuronyl activity. Kinetic analysis of purified oligo-ulvans incubated with the new enzyme showed that the full substrate specificity is attained by three subsites that preferentially bind anionic residues (sulfated rhamnose, glucuronic/iduronic acid). The three-dimensional crystal structure of the native enzyme reveals that a trimeric organization is required for substrate binding and recognition at the +2 binding subsite. This novel unsaturated ß-glucuronyl hydrolase is part of a previously uncharacterized subgroup of GH105 members and exhibits only a very limited sequence similarity to known unsaturated ß-glucuronyl sequences previously found only in family GH88. Clan-O formed by families GH88 and GH105 was singular in the fact that it covered families acting on both axial and equatorial glycosidic linkages, respectively. The overall comparison of active site structures between enzymes from these two families highlights how that within family GH105, and unlike for classical glycoside hydrolysis, the hydrolysis of vinyl ether groups from unsaturated saccharides occurs independently of the α or ß configuration of the cleaved linkage.

Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection.

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 µg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage.

Aqueous phase recycling: impact on microalgal lipid accumulation and biomass quality.

The potential success of microalgal biofuels greatly depends on the sustainability of the chosen pathway to produce them. Hydrothermal liquefaction (HTL) is a promising route to convert wet algal biomass into biocrude. Recycling the resulting HTL aqueous phase (AP) aims not only to recover nutrients from this effluent but also to use it as a substrate to close the photosynthetic loop and produce algal biomass again and process this biomass again into new biocrude. With that purpose, the response to AP recycling of five Chlorellaceae strains was monitored over five cultivation cycles. After four successive cycles of dynamic growth under nutrient-replete conditions, the microalgae were cultivated for a prolonged fifth cycle of 18 days in order to assess the impact of the AP on lipid and biomass accumulation under nutrient-limited conditions. Using AP as a substrate reduced the demand for external sources of N, S, and P while producing a significant amount of biomass (2.95-4.27 g/L) among the strains, with a lipid content ranging from 16 to 36%. However, the presence of the AP resulted in biomass with suboptimal properties, as it slowed down the accumulation of lipids and thus reduced the overall energy content of the biomass in all strains. Although Chlorella vulgaris NIES 227 did not have the best growth on AP, it did maintain the best lipid productivity of all the tested strains. Understanding the impact of AP on microalgal cultivation is essential for further optimizing biofuel production via the HTL process.

Microalgae adaptation as a strategy to recycle the aqueous phase from hydrothermal liquefaction.

Hydrothermal liquefaction (HTL) produces bio-crude oil from wet algae along with an aqueous phase (AP). This effluent contains minerals that can be reused for cultivating new microalgae but whose utility remains limited due to the presence of inhibitors. Reduced photosynthetic performance, growth, and null lipid accumulation were observed in wild-type Chlorella vulgaris NIES 227 cultivated in AP (1/200). Adaptive laboratory evolution was studied by batch transfers and turbidostat mode. Both methods effectively counterbalanced AP toxicity and restored the fitness of the microalgae. After adaptation, a higher AP addition was achieved, from 1/600 to 1/200, without inhibition. As compared with the wild typein control medium (0.261 g/L/d), both adapted-strains maintained competitive productivity (0.310 and 0.258 g/L/d) of lipid-rich biomass (37 %-56 %). The improved tolerance of the adapted strains persisted after the removal of AP and under axenic conditions. Adaptive laboratory evolution is suggested for AP reutilization in the algae production process.

Ulvan lyases isolated from the Flavobacteria Persicivirga ulvanivorans are the first members of a new polysaccharide lyase family.

Ulvans are complex sulfated polysaccharides found in the cell walls of green algae belonging to the genus Ulva. These polysaccharides are composed of disaccharide repetition moieties made up of sulfated rhamnose linked to either glucuronic acid, iduronic acid, or xylose. Two ulvan lyases of 30 and 46 kDa were purified from the culture supernatant of Persicivirga ulvanivorans. Based on peptide sequencing, the gene encoding the 46-kDa ulvan lyase was cloned. Sequence analysis revealed that the protein is modular and possesses a catalytic module similar to that of the 30-kDa ulvan lyase along with a module of unknown function. The ulvan-degrading function of the gene was confirmed by expression of the catalytic module in a heterologous system. The gene encoding the catalytic module has no sequence homolog in sequence databases and is likely to be the first member of a novel polysaccharide lyase family. Analysis of degradation products showed that both the 30- and 46-kDa ulvan lyases are endolytic and cleave the glycosidic bond between the sulfated rhamnose and a glucuronic or iduronic acid.

Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes.

BACKGROUND: The robust model cyanobacterium Synechocystis PCC 6803 is increasingly explored for its potential to use solar energy, water and atmospheric CO2 for the carbon-neutral production of terpenes, the high-value chemicals that can be used for the production of drugs, flavors, fragrances and biofuels. However, as terpenes are chemically diverse, it is extremely difficult to predict whether Synechocystis is a suitable chassis for the photosynthetic production of various terpenes or only a few of them. RESULTS: We have performed the first-time engineering and comparative analysis of the best-studied cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of five chemically diverse high-value terpenes: two monoterpenes (C10H16) limonene (cyclic molecule) and pinene (bicyclic), and three sesquiterpenes (C15H24) bisabolene (cyclic), farnesene (linear) and santalene (cyclic). All terpene producers appeared to grow well and to be genetically stable, as shown by the absence of changes in their production levels during the 5-9-month periods of their sub-cultivation under photoautotrophic conditions). We also found that Synechocystis PCC 6803 can efficiently and stably produce farnesene and santalene, which had never been produced before by this model organism or any other cyanobacteria, respectively. Similar production levels were observed for cells growing on nitrate (the standard nitrogen source for cyanobacteria) or urea (cheaper than nitrate). Furthermore, higher levels of farnesene were produced by cloning the heterologous farnesene synthase gene in a RSF1010-derived replicating plasmid as compared to the well-used slr0168 neutral cloning site of the chromosome. CONCLUSIONS:  Altogether, the present results indicate that Synechocystis PCC 6803 is better suited to produce sesquiterpenes (particularly farnesene, the most highly produced terpene of this study) than monoterpenes (especially pinene).

Persicivirga ulvanivorans sp. nov., a marine member of the family Flavobacteriaceae that degrades ulvan from green algae.

A rod shaped, Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, non-gliding bacterium, designated strain PLR(T), was isolated from faeces of the mollusc Aplysia punctata (Mollusca, Gastropoda) that had been fed with green algae belonging to the genus Ulva. The novel strain was able to degrade ulvan, a polysaccharide extracted from green algae (Chlorophyta, Ulvophyceae). The taxonomic position of strain PLR(T) was investigated by using a polyphasic approach. Strain PLR(T) was dark orange, oxidase-positive, catalase-positive and grew optimally at 25 °C, at pH 7.5 and in the presence of 2.5 % (w/v) NaCl with an oxidative metabolism using oxygen as the electron acceptor. Nitrate could not be used as the electron acceptor. Strain PLR(T) had a Chargaff's coefficient (DNA G+C content) of 35.3 mol%. Phylogenetic analysis based on the sequence of the 16S rRNA gene placed the novel strain in the family Flavobacteriaceae (phylum 'Bacteroidetes'), within a clade comprising Stenothermobacter spongiae, Nonlabens tegetincola, Sandarakinotalea sediminis, Persicivirga xylanidelens and Persicivirga dokdonensis. The closest neighbours of strain PLR(T) were P. xylanidelens and P. dokdonensis, sharing 95.2 and 95.5 % 16S rRNA gene sequence similarity, respectively. Phylogenetic inference and differential phenotypic characteristics demonstrated that strain PLR(T) represents a novel species of the genus Persicivirga, for which the name Persicivirga ulvanivorans sp. nov. is proposed. The type strain is PLR(T) ( = CIP 110082(T) = DSM 22727(T)).

Oligomannuronates from seaweeds as renewable sources for the development of green surfactants.

The development of surfactants based on natural renewable resources is a concept that is gaining recognition in detergents, cosmetics, and green chemistry. This new class of biodegradable and biocompatible products is a response to the increasing consumer demand for products that are both "greener", milder, and more efficient. In order to achieve these objectives, it is necessary to use renewable low-cost biomass that is available in large quantities and to design molecular structures through green processes that show improved performance, favorable ecotoxicological properties and reduced environmental impact. Within this context, marine algae represent a rich source of complex polysaccharides and oligosaccharides with innovative structures and functional properties that may find applications as starting materials for the development of green surfactants or cosmetic actives. Thus, we have developed original surfactants based on mannuronate moieties derived from alginates (cell-wall polyuronic acids from brown seaweeds) and fatty hydrocarbon chains derived from vegetable resources. Controlled chemical and/or enzymatic depolymerizations of the algal polysaccharides give saturated and/or unsaturated functional oligomannuronates. Clean chemical processes allow the efficient transformation of the oligomers into neutral or anionic amphiphilic molecules. These materials represent a new class of surface-active agents with promising foaming/emulsifying properties.

Recent Advances in the Photoautotrophic Metabolism of Cyanobacteria: Biotechnological Implications.

Cyanobacteria constitute the only phylum of oxygen-evolving photosynthetic prokaryotes that shaped the oxygenic atmosphere of our planet. Over time, cyanobacteria have evolved as a widely diverse group of organisms that have colonized most aquatic and soil ecosystems of our planet and constitute a large proportion of the biomass that sustains the biosphere. Cyanobacteria synthesize a vast array of biologically active metabolites that are of great interest for human health and industry, and several model cyanobacteria can be genetically manipulated. Hence, cyanobacteria are regarded as promising microbial factories for the production of chemicals from highly abundant natural resources, e.g., solar energy, CO2, minerals, and waters, eventually coupled to wastewater treatment to save costs. In this review, we summarize new important discoveries on the plasticity of the photoautotrophic metabolism of cyanobacteria, emphasizing the coordinated partitioning of carbon and nitrogen towards growth or compound storage, and the importance of these processes for biotechnological perspectives. We also emphasize the importance of redox regulation (including glutathionylation) on these processes, a subject which has often been overlooked.

A Genetic Toolbox for the New Model Cyanobacterium Cyanothece PCC 7425: A Case Study for the Photosynthetic Production of Limonene.

Cyanobacteria, the largest phylum of prokaryotes, perform oxygenic photosynthesis and are regarded as the ancestors of the plant chloroplast and the purveyors of the oxygen and biomass that shaped the biosphere. Nowadays, cyanobacteria are attracting a growing interest in being able to use solar energy, H2O, CO2 and minerals to produce biotechnologically interesting chemicals. This often requires the introduction and expression of heterologous genes encoding the enzymes that are not present in natural cyanobacteria. However, only a handful of model strains with a well-established genetic system are being studied so far, leaving the vast biodiversity of cyanobacteria poorly understood and exploited. In this study, we focused on the robust unicellular cyanobacterium Cyanothece PCC 7425 that has many interesting attributes, such as large cell size; capacity to fix atmospheric nitrogen (under anaerobiosis) and to grow not only on nitrate but also on urea (a frequent pollutant) as the sole nitrogen source; capacity to form CO2-sequestrating intracellular calcium carbonate granules and to produce various biotechnologically interesting products. We demonstrate for the first time that RSF1010-derived plasmid vectors can be used for promoter analysis, as well as constitutive or temperature-controlled overproduction of proteins and analysis of their sub-cellular localization in Cyanothece PCC 7425. These findings are important because no gene manipulation system had been developed for Cyanothece PCC 7425, yet, handicapping its potential to serve as a model host. Furthermore, using this toolbox, we engineered Cyanothece PCC 7425 to produce the high-value terpene, limonene which has applications in biofuels, bioplastics, cosmetics, food and pharmaceutical industries. This is the first report of the engineering of a Cyanothece strain for the production of a chemical and the first demonstration that terpene can be produced by an engineered cyanobacterium growing on urea as the sole nitrogen source.

Microalgae starch-based bioplastics: Screening of ten strains and plasticization of unfractionated microalgae by extrusion.

Microalgae were considered in this work as a new resource for developing starch-based bioplastics. Ten green microalgae strains were screened at lab-scale for their ability to produce starch. A long run (800 h) two-stage accumulation strategy was designed with successive cultivation in sulfur-replete, then sulfur-depleted medium in autotrophic conditions. Starch content was assessed on cell lysate by enzymatic digestion of extracted starch into glucose. Chlamydomonas reinhardtii 11-32A strain was selected as it displayed a maximum starch-to-biomass ratio of 49%w/w, 460 h after being switched to a sulfur-deprived medium. Small-scale pilot production (30 L tubular photobioreactor) with C. reinhardtii 11-32A yielded sufficient biomass quantity to investigate its direct plasticization with glycerol in a twin-screw extruder. Microstructural characterization confirmed the ability for starch-enriched microalgae to be homogeneously plasticized, and hence the possibility to use microalgae as a new platform for the development of bioplastics.

Giant dendrimer-like particles from nanolatexes.

A straightforward grafting of a polycationic phosphorus-containing dendritic shell onto polystyrene nanoparticles leads to dendronized nanoparticles showing unique behavior.

Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts.

Polysaccharides are the most abundant and the most diverse renewable materials found on earth. Due to the stereochemical variability of carbohydrates, polysaccharide-degrading enzymes - i.e. glycoside hydrolases and polysaccharide lyases - are essential tools for resolving the structure of these complex macromolecules. The exponential increase of genomic and metagenomic data contrasts sharply with the low number of proteins that have ascribed functions. To help fill this gap, we designed and implemented a medium-throughput profiling method to screen for polysaccharide-degrading enzymes in crude bacterial extracts. Our strategy was based on a series of filtrations, which are absolutely necessary to eliminate any reducing sugars not directly generated by enzyme degradation. In contrast with other protocols already available in the literature, our method can be applied to any panel of polysaccharides having known and unknown structures because no chemical modifications are required. We applied this approach to screen for enzymes that occur in Pseudoalteromonas carrageenovora grown in two culture conditions.

Ultrastructure of ulvan: a polysaccharide from green seaweeds.

Ultrastructural analysis of the gel forming green seaweed sulfated polysaccharide ulvan revealed a spherical-based morphology (10-18 nm diameter) more or less aggregated in aqueous solution. At pH 13 in TBAOH (tetrabutyl ammonium hydroxyde) or NaOH, ulvan formed an open gel-like structure or a continuous film by fusion or coalescence of bead-like structures, while in acidic pH conditions, ulvan appeared as dispersed beads. Low concentrations of sodium chloride, copper or boric acid induced the formation of aggregates. These results highlight the hydrophobic and aggregative behavior of ulvan that are discussed in regard to the peculiar gel formation and the low intrinsic viscosity of the polysaccharide in aqueous solution. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 652-664, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

SEASONAL VARIABILITY OF PHYSICOCHEMICAL AND RHEOLOGICAL PROPERTIES OF ULVAN IN TWO ULVA SPECIES (CHLOROPHYTA) FROM THE BRITTANY COAST(1).

The seasonal variability in the extraction yield, physicochemical characteristics, and rheological properties of ulvan from two Ulva species contributing to Brittany "green tides" has been studied. These seaweeds were collected in the water column for Ulva armoricana Dion, de Reviers et Coat and on hard substrata for Ulva rotundata Bliding. The maximum ulvan extraction efficiency was not related to the maximum ulvan content in the seaweeds, but with the active growth period of the seaweeds. Ulvan chemical structure, macromolecular characteristics, and rheological properties were affected by both species and seasons. The proportion of high-molecular-weight ulvan was the major factor positively correlated with the gelling properties. Characteristics of ulvan from U. rotundata subjected to tides were more affected by seasons than ulvan from U. armoricana living in a more constant environment. These results point to several useful recommendations concerning Ulva sp. biomass collected with regard to ulvan characteristics and uses.