MICROALGAE BIOTECHNOLOGY AND MICROBIOLOGY PDF
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Download Microalgae Biotechnology And Microbiology Cambridge Studies In. Biotechnology free pdf, Download Microalgae Biotechnology And Microbiology. Microalgae: Biotechnology, Microbiology And Energy - Gbv contents preface vii Microalgae Biotechnology And Microbiology Pdf - dovolena-na-lodi.info microalgae . The authors have adopted an integrated approach, combining theory and practice in an illustrative manner with a large number of black and white photographs.
These benefits contribute to the economic viability of microalgal production [ 7 , 8 ]. Bioactive compounds are physiologically active substances with functional properties in the human body. There is great enthusiasm for the development and manufacture of various biocompounds that can potentially be used as functional ingredients, such as carotenoids, phycocyanins, polyphenols, fatty acids, and polyunsaturated compounds [ 16 ]. An interest in the production of bioactive compounds from natural sources has recently emerged, driven by a growing number of scientific studies that demonstrate the beneficial effects of these compounds on health [ 80 ].
Natural products are important in the search for new pharmacologically active compounds. In general, they play a role in drug discovery for the treatment of human diseases [ 86 ]. Many clinically viable and commercially available drugs with antitumor and antiinfective activity originated as natural products.
Microalgae are a natural source of interesting biocompounds. Microalgae are known to produce various therapeutically effective biocompounds that can be obtained from the biomass or released extracellularly into the medium [ 11 ]. These microorganisms contain many bioactive compounds, such as proteins, polysaccharides, lipids, vitamins, enzymes, sterols, and other high-value compounds with pharmaceutical and nutritional importance that can be employed for commercial use [ 13 ].
Oxidative damage caused by reactive oxygen species to lipids, proteins, and nucleic acids can cause many chronic diseases such as heart disease, atherosclerosis, cancer, and aging. Epidemiological studies have demonstrated an inverse association between the intake of fruits and vegetables and mortality from diseases such as cancer.
This phenomenon can be attributed to the antioxidant activity of these foods [ 87 ]. Microalgal biomass is considered a rich natural source of antioxidants, with potential applications in food, cosmetics, and medicine [ 87 ]. Antioxidant compounds, such as dimethylsulfoniopropionate and mycosporine amino acids, were isolated from microalgae and are potent chemical blockers of UV radiation [ 88 ].
In addition to these compounds, pigments, lipids, and polysaccharides with antioxidant activity can also be found in microalgal biomass. Carotenoids and phycocyanins are the pigments most used in scientific research.
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C-phycocyanin C-PC is a blue photosynthetic pigment that belongs to the group of phycobiliproteins found in large quantities in the cyanobacteria, Rhodophyta, and Cryptophyte [ 89 ]. Phycocyanin has applications as a nutrient and natural food colorants and cosmetics.
It is usually extracted from the biomass of Spirulina [ 90 ] and Porphyridium cruentum [ 91 ] and Synechococcus [ 89 ]. These compounds have application in the food and pharmaceutical industries because of their antioxidant properties and pigmentation ability.
In microalgal metabolism, they protect photosynthetic tissues against damage caused by light and oxygen [ 92 ]. Polysaccharides represent a class of high value-added components with applications in food, cosmetics, fabrics, stabilizers, emulsifiers, and medicine [ 94 ]. Microalgal polysaccharides contain sulphate esters, are referred to as sulfated polysaccharides, and possess unique medical applications. The basic mechanism of therapeutic action is based on the stimulation of macrophages and modulation.
The biological activity of sulfur polysaccharides is linked to their sugar composition, position, and degree of sulfation [ 95 ].
Among the microalgae capable of producing these compounds are Chlorella vulgaris , Scenedesmus quadricauda [ 96 ], and Porphyridium sp. The importance of discovering new compounds with antimicrobial activity is driven by the development of antibiotic resistance in humans due to constant clinical use of antibiotics. Microalgae are an important source of antibiotics with a broad and efficient antibacterial activity [ 11 ]. The antimicrobial activity of these microorganisms is due to the ability to synthesize compounds, such as fatty acids, acrylic acids, halogenated aliphatic compounds, terpenoids, sterols, sulfur-containing heterocyclic compounds, carbohydrates, acetogenins, and phenols [ 98 ].
The antimicrobial activity of extracts from microalgae is related to its lipid composition. The mechanism of action of fatty acids affects various structures in microorganisms; however cell membranes are the most impacted.
Membrane damage most likely leads to a loss of internal substances from the cells, and the entry of harmful components reduces nutrient absorption, in addition to inhibiting cellular respiration.
The ability of fatty acids to interfere with bacterial growth depends on both their chain length and the degree of unsaturation. Fatty acids with more than 10 carbon atoms apparently induce lysis of bacterial protoplasts [ 99 ]. Microbial polysaccharides and other biological compounds have antiviral and antimicrobial action. Microalgae produce extracellular sulfated polysaccharide EPS with acidic characteristics that has a potential as a therapeutic agent [ ].
Highly sulfated antiviral polysaccharides from several species of microalgae consist mainly of xylose, glucose, and galactose. The EPS sulfate groups determine some characteristics of polysaccharides; it has been found that higher sulphate contents induced higher antiviral activities [ 94 , ]. The inhibitory effect of polysaccharides of microalgal origin is due to viral interactions or positive charges on the cell surface, thereby preventing penetration of the virus into host cells [ 99 ].
The cyanobacterium Spirulina Arthrospira can produce sulfated polysaccharides that have already found applications as antiviral agents, both in vivo and in vitro [ ]. Eukaryotic microalgae, such as Chlorella sp. Some studies have reported that sulfated polysaccharides derived from microalgae inhibit viral infection, such as encephalomyocarditis virus, Herpes simplex virus types 1 and 2 HSV1, HSV2 , human immunodeficiency virus HIV , hemorrhagic septicemia in salmonid virus, swine fever virus, and varicella virus [ 99 , ].
Carrageenan is a sulfated polysaccharide that can directly bind to human papillomavirus to inhibit not only the viral adsorption process but also the input and subsequent process of the uncoating of the virus [ ]. Inflammation is an immediate reaction to a cell or tissue injury caused by noxious stimuli, such as toxins and pathogens.
In this situation, the body recognizes the agents responsible for the attack and attempts to neutralize them as quickly as possible. Inflammation causes redness, swelling, heat, and pain, usually located at the site of infection [ ].
Ingestion of anti-inflammatory compounds enhances the body's immune response and helps to prevent disease and aids the healing process. Microalgae produce several anti-inflammatory compounds in their biomass that may exert a protective function in the body when consumed as food or used as pharmaceuticals and cosmetics. Because of its anti-inflammatory capabilities, microalgal biomass is being considered for applications in tissue engineering for the development of scaffolds, for use in reconstitution of organs and tissues [ , ].
This is an important application for humans, especially in patients with burns in which the skin was completely lost [ ]. Among the most important microalgal compounds with such properties are long-chain polyunsaturated fatty acids PUFAs [ , ], sulfurized polysaccharides [ ], and pigments [ ].
Macrophages are able to regulate several innate responses and secrete cytokines and chemocytokines that serve as signals for immune and inflammatory molecular reactions [ ]. Sulfur polysaccharides with anti-inflammatory activity can be applied in skin treatments inhibiting the migration and adhesion of polymorphonuclear leukocytes [ ]. Ryckebosch et al. Among the pigments with anti-inflammatory activity, fucoxanthin carotenoid found in diatoms [ , ] is capable of stimulating apoptosis in human cancer cells [ ].
A phycocyanin, found in cyanobacteria, has an anti-inflammatory activity that occurs through the inhibition of histamine release [ , ]. Consequently, this damage can lead to several syndromes, such as cardiovascular disease, some cancers, and the degenerative diseases of aging [ ]. Chronic age-related diseases involve oxidative stress and inflammation and their consequences.
Chronic inflammation plays a significant role in the mediation of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, acquired immunodeficiency syndrome AIDS , and dementia complex [ 77 ]. Vitamin E has preventive effects for many diseases, such as atherosclerosis and heart disease, as well as neurodegenerative diseases, such as multiple sclerosis [ 77 ].
Carotenoids have great potential benefits to human health, including the treatment of degenerative diseases, such as macular degeneration and cataract development.
Microalgal mass culture systems and methods: Their limitation and potential
These compounds act as antioxidants, reducing oxidative damage by ROS. Studies indicated that increased intake of phenols decreased the occurrence of degenerative diseases. Phenolic compounds from microalgae with the potential to fight free radicals have been reported [ ]. Lutein is effective against various diseases, including cataracts and macular degeneration, and in the early stages of atherosclerosis.
Extracts of Chlorella sp. It was also reported that lutein extracted from Chlorella reduced the incidence of cancer. Likewise, carotenoids extracted from Chlorella ellipsoidea and Chlorella vulgaris inhibited the growth of colon cancer [ ].
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A lycopene extracted from the microalgae Chlorella marina significantly reduced the proliferation of prostate cancer in mice [ ]. This compound also reduced total cholesterol and low-density lipoprotein LDL levels [ ] and improved rheumatoid arthritis [ ]. Low plasma levels of lutein have also been associated with an increased tendency of myocardial infarction, whereas high intake of lutein was related to a decreased risk of stroke.
Macular degeneration, the leading cause of irreversible vision loss, has also been associated with very low consumption of lutein and zeaxanthin [ ]. Scientific findings indicate astaxanthin for multimodal intervention for many forms of degenerative diseases, including cardiovascular diseases, cancer, metabolic syndrome, cognitive impairment, age-related immune dysfunction, stomach and ocular diseases macular degeneration, cataract, glaucoma, diabetic retinopathy, and retinitis pigmentosa , and skin damage [ ].
High levels of lycopene in plasma and tissues were inversely related to coronary heart disease, myocardial infarction, and the risk of atherosclerosis [ ]. The importance of microalgae as sources of functional ingredients has been recognized because of their beneficial health effects.
Natural pigments are valuable sources of bioactive compounds. These pigments have various beneficial biological activities such as antioxidant, anticancer, anti-inflammatory, antiobesity, antiangiogenic, and neuroprotective action and are indicated for the treatment or prevention of several chronic diseases [ 77 ].
The antioxidant potential of carotenoid pigments and their ability to prevent cancer, aging, atherosclerosis, coronary heart disease, and degenerative diseases have been described. Astaxanthin is linked to many health benefits such as protection against lipid peroxidation, age-related macular degeneration, reduced atherosclerosis, and an increased immune response [ ].
Fucoxanthin is considered as a promising dietary and weight loss supplement and for the treatment of obesity. Clinical studies by Abidov et al. Furthermore, fucoxanthin may be useful for the prevention of bone diseases such as osteoporosis and rheumatoid arthritis. It has also been reported to be effective for the therapeutic treatment of diabetic diseases, suppressing insulin and hyperglycemia [ 77 ].
Microalgae proteins are of great interest as a source of bioactive peptides due to their therapeutic potential in the treatment of various diseases [ 7 ]. Proteins, peptides, and amino acids have functions that contribute to health benefits. These compounds can include growth factors, hormones, and immunomodulators and can help to replace damaged tissues, in addition to providing nutritional benefits.
Microalgae, such as Chlorella and Spirulina Arthrospira , may be used as nutraceuticals or included in functional foods to prevent diseases and damage to cells and tissues due to their rich protein content and amino acid profile [ ]. The antimicrobial action of certain enzymes e. Studies of the health effects of lysozyme have been reported in the microalgae Spirulina platensis [ ], Chlorella [ ], and Dunaliella salina [ ].
Other proteins can also increase the production of cholecystokinin involved in appetite suppression and the reduction of LDL-cholesterol. Protein peptides from Chlorella have a potential as dietary supplements for the prevention of oxidative stress-related diseases, such as atherosclerosis, coronary heart disease, and cancer [ 39 ].
Linoleic and linolenic acids are essential nutrients for the immune system and other related tissue regeneration processes. Linoleic acid is also used for the treatment of hyperplasia of the skin [ ].
DHA and EPA showed the ability to reduce problems associated with strokes and arthritis, besides reducing hypertension, lipid content a decrease in triglycerides and an increase of HDL and acting as anti-inflammatory agents.
DHA is also important in the development and function of the nervous system.
Furthermore, ARA and EPA are platelet aggregators, vasoconstrictors, and vasodilators and have antiaggregative action on the endothelium, as well as chemostatic activity in neutrophils [ ]. Other lipid compounds with interesting bioactive properties are the microalgal sterols. Phytosterols have demonstrated reduction of total cholesterol LDL in humans by inhibiting its absorption from the intestine [ 50 ]. Polysaccharides can be considered as dietary fibers associated with different physiological effects.
Insoluble fiber cellulose, hemicellulose, and lignin mainly promotes the movement of material through the digestive system, thereby improving laxation and increasing satiety. They can also be considered as prebiotics because they promote the growth of gut microflora, including probiotic species. The proven ability of microalgae to produce bioactive compounds places these microorganisms in the biotechnological spotlight for applications in various areas of study, especially in the life sciences.
The production of microalgal metabolites, which stimulate defense mechanisms in the human body, has spurred intense study of the application of microalgal biomass in various foods and pharmacological and medical products. There is obviously a need for further study of the identified compounds and their activities in the treatment and prevention of various diseases, in addition to an ongoing search for other, as yet undetected, metabolites.
The authors declare that there is no conflict of interests regarding the publication of this paper. National Center for Biotechnology Information , U. Journal List Biomed Res Int v. Biomed Res Int. Published online Aug 3. Author information Article notes Copyright and License information Disclaimer. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC. Abstract Microalgae are microorganisms that have different morphological, physiological, and genetic traits that confer the ability to produce different biologically active metabolites.
Microalgae with Potential for Obtaining Bioactive Compounds Microalgae are a group of heterogeneous microorganisms that have a great biodiversity of colors, shapes, and cell characteristics, and their manipulation is encompassed by the field of marine biotechnology.
Table 1 Principal bioactive compounds extracted from microalgae. Microalgae Bioactive compounds Reference Spirulina sp. Cryptophycin [ 81 ].
Open in a separate window. Spirulina Spirulina Arthrospira is prokaryotic cyanobacteria Figure 1 that belongs to Cyanophyta, which arose more than 3 million years ago, forming the current oxygen atmosphere, and has been important in the regulation of the planetary biosphere [ 21 ]. Figure 1. Table 2 Bioactive compounds extracted from Spirulina genus.
C-phycocyanin Allophycocyanin Nostoc Nostoc is an edible microalga that belongs to the Nostocaceae group Cyanophyta that forms spherical colonies that link together as filaments. Figure 2. Table 3 Bioactive compounds extracted from the Nostoc genus. Phycocyanin Chlorella Spirulina and Chlorella represent the majority of the microalgal biomass market, with an annual production of 3, and 4, tons, respectively [ 38 ].
Table 4 Bioactive compounds extracted from the microalgae of the Chlorella genus. Dunaliella Dunaliella is a green unicellular halotolerant microalga that belongs to the Chlorophyceae group Figure 4.
Figure 4. Table 5 Bioactive compounds extracted from the microalgae of the Dunaliella genus. Cultivation Conditions The conditions for microalgal cultivation are important factors that influence the metabolism of these microorganisms, thus directing the synthesis of specific compounds of interest.
Bioreactors Microalgae have attracted much interest for production of bioactive compounds, and in order to grow and tap the potentials of algae, efficient photobioreactors are required. Nutrients The metabolism of microalgae can be autotrophic or heterotrophic. Advantages of Using Microalgae to Obtain Bioactive Compounds Microalgae are important sources of bioactive natural substances. Bioactive Compounds Bioactive compounds are physiologically active substances with functional properties in the human body.
Compounds with Antioxidant Function Oxidative damage caused by reactive oxygen species to lipids, proteins, and nucleic acids can cause many chronic diseases such as heart disease, atherosclerosis, cancer, and aging.
Compounds with Antimicrobial Activity The importance of discovering new compounds with antimicrobial activity is driven by the development of antibiotic resistance in humans due to constant clinical use of antibiotics.
Compounds with Anti-Inflammatory Action Inflammation is an immediate reaction to a cell or tissue injury caused by noxious stimuli, such as toxins and pathogens. Compounds with Health Promoting Function The importance of microalgae as sources of functional ingredients has been recognized because of their beneficial health effects.
Conclusion The proven ability of microalgae to produce bioactive compounds places these microorganisms in the biotechnological spotlight for applications in various areas of study, especially in the life sciences. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
References 1. Ferreira S. El Gamal A. Biological importance of marine algae. Saudi Pharmaceutical Journal. Moreno-Garrido I. Microalgae immobilization: Bioresource Technology. Volk R. Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth.
Microbiological Research. A newly developed assay for the quantitative determination of antimicrobial anticyanobacterial activity of both hydrophilic and lipophilic test compounds without any restriction. Smee D. Treatment of influenza A H1N1 virus infections in mice and ferrets with cyanovirin-N. Antiviral Research. Benefits of using algae as natural sources of functional ingredients.
Journal of the Science of Food and Agriculture. Markou G. Microalgae for high-value compounds and biofuels production: Biotechnology Advances. Harun R. Bioprocess engineering of microalgae to produce a variety of consumer products. Renewable and Sustainable Energy Reviews. Kolympiris C. Public funds and local biotechnology firm creation. Research Policy.
Commercial production of microalgae in the Asia-Pacific rim
Bhagavathy S. Green algae Chlorococcum humicola -a new source of bioactive compounds with antimicrobial activity. Asian Pacific Journal of Tropical Biomedicine. Palavra A. Supercritical carbon dioxide extraction of bioactive compounds from microalgae and volatile oils from aromatic plants. Journal of Supercritical Fluids.
Priyadarshani I. Commercial and industrial applications of micro algae—a review. Journal of Algal Biomass Utilization. Blunt J. Marine natural products. Natural Product Reports. Mayer A. Marine pharmacology in Comparative Biochemistry and Physiology Part C: Plaza M. Screening for bioactive compounds from algae. Journal of Pharmaceutical and Biomedical Analysis. Pressurized fluid extraction of bioactive compounds from Phormidium species.
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Carvalho L. Biologically active compounds from cyano bacteria extracts: Brazilian Journal of Pharmacognosy. Nobre B. Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. European Food Research and Technology. Mendes R. Food Chemistry. Romano I.
Lipid profile: Costa J. Microalgae for food production. Soccol C. Fermentation Process Engineering in the Food Industry. Harvey's four divisions are: red algae Rhodospermae , brown algae Melanospermae , green algae Chlorospermae , and Diatomaceae. Unlike macroalgae , which were clearly viewed as plants, microalgae were frequently considered animals because they are often motile.
Valuable products from biotechnology of microalgae
Throughout the 20th century, most classifications treated the following groups as divisions or classes of algae: cyanophytes , rhodophytes , chrysophytes , xanthophytes , bacillariophytes , phaeophytes , pyrrhophytes cryptophytes and dinophytes , euglenophytes , and chlorophytes. Later, many new groups were discovered e. With the abandonment of plant-animal dichotomous classification, most groups of algae sometimes all were included in Protista , later also abandoned in favour of Eukaryota.
However, as a legacy of the older plant life scheme, some groups that were also treated as protozoans in the past still have duplicated classifications see ambiregnal protists. Some parasitic algae e. In other cases, some groups were originally characterized as parasitic algae e. Furthermore, groups like the apicomplexans are also parasites derived from ancestors that possessed plastids, but are not included in any group traditionally seen as algae.
Relationship to land plants[ edit ] The first land plants probably evolved from shallow freshwater charophyte algae much like Chara almost million years ago. These probably had an isomorphic alternation of generations and were probably filamentous. Fossils of isolated land plant spores suggest land plants may have been around as long as million years ago.
The only groups to exhibit three-dimensional multicellular thalli are the reds and browns , and some chlorophytes. Some of the more common organizational levels, more than one of which may occur in the lifecycle of a species, are Colonial : small, regular groups of motile cells Capsoid: individual non-motile cells embedded in mucilage Coccoid: individual non-motile cells with cell walls Palmelloid: nonmotile cells embedded in mucilage Filamentous: a string of nonmotile cells connected together, sometimes branching Parenchymatous: cells forming a thallus with partial differentiation of tissues In three lines, even higher levels of organization have been reached, with full tissue differentiation.
The innovation that defines these nonalgal plants is the presence of female reproductive organs with protective cell layers that protect the zygote and developing embryo. Hence, the land plants are referred to as the Embryophytes. Physiology[ edit ] Many algae, particularly members of the Characeae ,  have served as model experimental organisms to understand the mechanisms of the water permeability of membranes, osmoregulation , turgor regulation , salt tolerance , cytoplasmic streaming , and the generation of action potentials.
Phytohormones are found not only in higher plants, but in algae, too. In these symbioses, the algae supply photosynthates organic substances to the host organism providing protection to the algal cells. The host organism derives some or all of its energy requirements from the algae. Diverse algal genera such as Nannochloropsis, Dunaliella, Chaetoceros, Botryococcus, Scenedesmus and Pseudochlorococcum are known to accumulate high amount of neutral lipids [ 38 — 40 ].
The metabolic pathways of algal strains are able to produce 16—20 carbon fatty acids as precursors for the production of biodiesel. Nobre et al. In a recent study, a high-lipid-producing microalga, namely Euglena sanguinea was investigated for biodiesel production [ 41 ].
The saturated fatty acids C, C, C, C and unsaturated fatty acids C in the biodiesel confirmed that these could be potentially used in automobiles without any considerable transition in engine design. Here, systems biology, especially flux analysis, can provide an effective means of prediction by tracking the carbon flux during lipid accumulation, carbon fixation and growth altogether. The enzymes that can be targeted to enhance growth and carbon fixation can be determined from enzyme flux control coefficient data of Calvin cycle enzymes [ 42 ].
Likewise, the targets involved in lipid metabolism can also be found by 13C metabolic flux data and subsequent metabolic map derived from oleaginous algae. These flux data reveal which enzymes and the pathways they regulate are rate limiting and exert significant control over the larger metabolism [ 43 , 44 ].
These developments would facilitate to increase the yield, concurrent with an economical algal biodiesel production in the near future. In addition to biofuels, microalgae are feedstock to several other high-value products such as vitamins, pigments, proteins, carbohydrates, amino acids, antioxidants, high-value long-chain polyunsaturated fatty acids PUFAs and biofertilizers.
Natural microalgal pigments such as carotenoids, chlorophylls and phycobiliproteins serve as precursors of vitamins in food, pharmaceutical industries, cosmetics and coloring agents [ 45 ]. Several studies are focussing on microalgal genes encoding enzymes, which are involved in high-value carotenoid synthesis.
Microalgae such as C. In another study, C. Microalgae also serve as potential expression systems for the synthesis of biopolymers such as polyhydroxybutyrate, which is a key precursor for the synthesis of biodegradable plastics. In a recent study, an ATP hydrolysis-based driving force module was engineered into Synechococcus elongatus PCC to produce 3-hydroxybutyrate [ 48 ].
The strain which was engineered by having a provision for a reversible outlet for excessive carbon flux was capable of producing significantly high amounts of 3-hydroxybutyrate over the native strain.
Several microalgae secrete extracellular polymeric substances in their immediate living environment as a hydrated biofilm protective matrix [ 49 ]. These substances are known for high-value applications such as anti-inflammatories, antivirals, antioxidants, anticoagulants, biolubricants and drag reducers.
In a recent study, LEA has been used as a substrate for biomethanation through anaerobic processes [ 51 ]. This study showed that the rate of biogas production was comparatively higher in product-extracted algal samples lipid and protein extracted , whilst the cumulative methane production was higher for pretreated algae dried powdered algae and heat-treated algae.
LEA has also been used as raw material for butanol fermentation [ 53 ]. Bench-scale tests demonstrated that LEA could also be effectively converted to liquid fuel, mainly alkanes via hydrothermal liquefaction and upgrading processes such as via hydrotreating and hydrocracking. The overall energy efficiency on a higher heating value basis of this process was estimated to be Several microalgae with a high nutritional value and energy content are grown commercially as aquaculture feed.
Communicating Current Research and Technological Advances. Abstract Microalgae are microorganisms that have different morphological, physiological, and genetic traits that confer the ability to produce different biologically active metabolites.
To ensure a successful mating, the development and release of gametes is highly synchronized and regulated; pheromones may play a key role in these processes.
Steffens D. The basic mechanism of therapeutic action is based on the stimulation of macrophages and modulation.
Edisud; Semary N.
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