Cyanobacterial Physiology: From Fundamentals to Biotechnology

Cyanobacteria are ancient, primordial oxygenic phototrophs, and probably the progenitor of oxygen-evolving photosynthesis. They are a prolific source of natural products and metabolites and vitally important for environmental biology and biotechnology.

Cyanobacterial Physiology presents foundational knowledge alongside the most recent advances in cyanobacterial biology. The title examines the challenges of industrial application through an understanding of the basic molecular machinery of cyanobacteria. Sixteen chapters are organized into three sections. The first part covers basic cyanobacterial biology, emphasizing environmental biology such as photosynthesis, nitrogen fixation, circadian rhythm, and programmed cell death. The second part includes the chapters that discuss cyanobacterial extremophiles, adaptations, secondary metabolites, osmoprotectants, and toxins. The third part covers aspects of cyanobacterial application that are based on environmental biology. Leading scientists contribute chapters on cyanobacteria.

Cyanobacterial Physiology presents a comprehensive and vibrant solution for researchers and engineers in biotechnology interested in cyanobacteria and their applications. Topics include the cyanobacterial cell and fundamental physiological processes; the biotechnological potential of cyanobacteria with their versatile metabolism; and advanced applications of cyanobacterial products. At each stage the book is informed by basic and applied research.

• Examines industrial applications of cyanobacteria through their basic molecular machinery
• Presents foundational knowledge about cyanobacteria alongside the latest research
• Leading scientists present basic and applied research on cyanobacteria
• Covers cyanobacterial biology and applications in environmental biotechnology
• Give researchers and engineers a comprehensive solution for working with cyanobacteria in relation to environmental biology and biotechnology

• Language: English
• Publisher: Academic Press
• Release date: May 31, 2022
• ISBN: 9780323993869

Book preview

Preface

Hakuto Kageyama; Rungaroon Waditee-Sirisattha

Among prokaryotes, cyanobacteria are primordial oxygenic phototrophs, which have evolved from the progenitors of oxygen-evolving photosynthesis. Cyanobacteria are valuable and prolific sources of natural products and/or metabolites with intricate chemical structures and potent biological activities. The past and current knowledge concerning the physiology of cyanobacteria have been collected and presented in the book Cyanobacterial Physiology: From Fundamentals to Biotechnology. Since this book covers various topics from fundamental descriptions of cyanobacterial physiological phenomena to numerous biotechnological applications of cyanobacteria, it may serve as a key reference to all those working with cyanobacteria, whether in the laboratory, in the field, or in the private sector. We are confident that this book will be a valuable resource for understanding the basic molecular mechanisms of cyanobacteria and for addressing questions regarding the challenges of developing industrial applications of cyanobacteria.

This book consists of 3 parts with a total of 16 chapters. The chapters were written by several accomplished scientists, who have described the critical and major molecular mechanisms of cyanobacteria and cyanobacterial physiology.

In Part I, the fundamental and physiological characteristics of cyanobacteria are presented. In Chapter 1, Waditee-Sirisattha and Kageyama review cyanobacterial cells in terms of their fundamental morphology, physiology, ecological importance, and classification. They also discuss the emerging tools of omics and cyanobacterial databases. In Chapter 2, Katayama describes the basic process and the current understanding of cyanobacterial photosynthesis, which is a fundamental process that converts light energy into chemical energy. In Chapter 3, Fujita and Uesaka provide a detailed review of cyanobacterial nitrogen fixation from the perspective of the Oxygen Paradox. In addition to the basic molecular mechanisms, they discuss the applied aspects of cyanobacterial nitrogen-fixing ability. In Chapters 4 and 5, the cyanobacterial circadian clock is described. Terauchi and Onoue present an overview of the classical studies of the cyanobacterial circadian clock as well as the latest studies focusing on the phosphorylation of the KaiC protein and its ATPase activity. Ito reviews the relationships between the cyanobacterial circadian clock and temperature. In Chapter 6, Li and colleagues discuss cyanobacterial regulated cell death (RCD). They summarize the significance of cyanobacterial RCD and propose a scheme to classify the types of cyanobacterial cell death based on the involvement of caspase homologues.

Part II includes chapters that discuss cyanobacterial extremophiles, adaptations, secondary metabolites, osmoprotectants, and toxins. In Chapter 7, the key features of five extremophilic cyanobacterial groups are introduced. In addition, their environmental niches as well as the molecular mechanisms and metabolic machinery that contribute to their stress responses are comprehensively reviewed. Chapter 8 focuses on cyanobacterial multifunctional secondary metabolites, including mycosporine-like amino acids (MAAs) and scytonemins. This chapter also discusses the relevant properties and potential applications of these compounds. In Chapter 9, the basic features, biosynthetic pathways, and regulation of cyanobacterial osmoprotectant molecules, including glycine betaine, glucosylglycerol, glucosylglycerate, sucrose, and trehalose, as well as the rare potential osmoprotectant molecules, including homoserine betaine, glutamate betaine, proline, glycerol, and dimethylsulfoniopropionate, are described. In Chapters 10 and 11, Hu summarizes the most common bloom-forming cyanobacterial genus, Microcystis, which can deteriorate water quality by production and release of the potent liver toxin microcystin. In Chapter 10, the practical analytical methods, biosynthesis, and biofunctions of microcystin blooms are reviewed and discussed. In Chapter 11, the current knowledge regarding the detection, biosynthesis, and biofunction of MAAs from Microcystis is provided. In addition, the genetic markers of MAA-producing Microcystis are described.

In Part III, five chapters discuss various biotechnological applications of cyanobacteria. In Chapter 12, Bakku and Rakwal focus on cyanobacterial bioactive compounds, including lipids, phenols, alkaloids, pigments, vitamins, proteins, enzymes, and cytotoxins. They also describe their current as well as potential future applications for the next generation. In Chapter 13, Wang and colleagues discuss cyanobacterial synthetic biology. They summarize the current status of chemical production in cyanobacteria and highlight the importance of utilizing high-flux pathways to produce diverse chemicals at high concentrations. Moreover, they describe several key routes for improving the efficiency of cyanobacterial chemical production using a variety of systems and synthetic biology tools. In Chapter 14, Osanai and colleagues summarize various tools for (1) the genetic manipulation of cyanobacteria and (2) the characterization of recently isolated cyanobacterial metabolic enzymes. These tools may usher in an era of cyanobacterial synthetic biology. In Chapter 15, Narikawa and colleagues provide a review of various cyanobacterial photoreceptors that regulate specific light-acclimation processes. In addition, they describe the photosensory mechanisms of the cyanobacterial photoreceptors with a special emphasis on their biochemical, photochemical, and physiological characteristics. In Chapter 16, Honda reviews cyanobacterial carotenoids, including the potential use of cyanobacteria as carotenoid producers. The applications of various carotenoids, the biological activities exhibited by carotenoids, and the recent progress in carotenoid processing technology are further summarized.

We thank the authors of all the chapters for their kind contributions. We are also grateful to Dr. Glyn Jones, Ms. Tracy Tufaga, and the entire Elsevier team for their cooperation and assistance with the editorial process.

Editors

Part I

Fundamental and physiological characteristics of cyanobacteria

Chapter 1: Cyanobacterial cells

Rungaroon Waditee-Sirisatthaa,⁎; Hakuto Kageyamab,c,⁎    a Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

b Graduate School of Environmental and Human Sciences, Meijo University, Nagoya, Japan

c Department of Chemistry, Faculty of Science and Technology, Meijo University, Nagoya, Japan

Abstract

Cyanobacteria are a group of oxygen-evolving prokaryotes that occur ubiquitously on our planet. They occupy diverse habitats, in both aquatic and terrestrial ecosystems, including extreme environments, as well as form endolithic and endophytic microbial communities. Fossil evidence suggests that cyanobacteria were among the first organisms to inhabit Earth, dating back some 3.5 billion years, and have made great contributions to the planet. For example, cyanobacteria have played a key role in Earth's transition from an anaerobic to an aerobic environment, occupy a central position in nutrient-recycling processes, and represent a valuable source of natural products. In the first section of this chapter, we describe cyanobacterial cells, including the aspects of their fundamental morphology, physiology, and ecological importance. The second section presents a discussion of cyanobacterial classification, including classical taxonomy, modern molecular taxonomy, and polyphasic approaches. Finally, we discuss the emerging tools of omics and cyanobacterial databases.

Keywords

Cyanobacteria; Morphology; Polyphasic taxonomy; Nutrient recycling; Central metabolism; Natural products

Outline

1Introduction

2Structural components and morphology of cyanobacterial cells

2.1Structural components

2.2Genetic material

2.3Granules

2.4Specialized structures

2.5Morphology

2.6Reproduction

3Ecological importance

3.1Primary producers in food webs and use as food supplements

3.2Nitrogen fixing and nutrient recycling

3.3Cyanobacterial blooms and cyanotoxins

4Classical taxonomy, modern molecular phylogeny, and polyphasic approaches

5Emerging tools: Omics and cyanobacterial databases

6Conclusions

References

1: Introduction

The various forms of life on Earth exclusively comprise prokaryotes and eukaryotes, which are classified according to the organization of their cellular structures. Cyanobacteria are a group of fundamentally oxygen-evolving prokaryotes; they are considered to be important contributors to photosynthetic biomass production. One of the many interesting characteristics of cyanobacteria is their broad geographical distribution. They occur ubiquitously around the planet, inhabiting diverse habitats in both aquatic and terrestrial ecosystems, as well as forming endolithic and endophytic microbial communities. They can even be found thriving in extreme environments, such as hot springs, hypersaline lakes, deserts, and polar regions [1–3].

Fossil evidence suggests that cyanobacteria were among the first microorganisms to inhabit Earth, dating back 3.5 billion years to the Precambrian time [4]. As the oldest recorded fossils, cyanobacteria were centrally involved in the evolution of life by acting as oxygen accumulators. They have played a key role in Earth's transition from its early anaerobic state to its current aerobic condition [5,6]. In terms of their ecological functions, cyanobacteria form an important part of the base of the aquatic food chain, are involved in nutrient-recycling processes, and represent a valuable source of natural products [6], while some filamentous cyanobacteria possess the ability to fix atmospheric nitrogen [7]. Hence, cyanobacteria are important contributors to the biosphere and fulfill vital ecological functions.

Morphologically, cyanobacteria range from unicellular to filamentous types, with some species being colony formers. Certain filamentous cyanobacteria are capable of cell differentiation, enabling their cells to perform specific functions, thus indicating that multicellularity has evolved. This characteristic is unique among prokaryotes. Many cyanobacterial species, known as extremophiles, exhibit a remarkable flexibility and have adapted to a wide range of extreme environmental conditions [2,3]. A full review of cyanobacterial extremophiles and their characteristics can be found in Chapter 7.

In this chapter, we first discuss the structural components and morphology of cyanobacterial cells; then, we describe the ecological importance of cyanobacteria and the environmental services they provide; and, finally, we focus on the classification of cyanobacteria, with a discussion of classical morphology, modern molecular phylogeny, and polyphasic approaches, including an overview of cyanobacterial databases.

2: Structural components and morphology of cyanobacterial cells

Cyanobacterial cells are larger and more elaborate than bacterial cells. Typically, they display cell sizes in the range of 1–10 μm [8], which is one-tenth to one-twentieth the size of eukaryotic cells. Living forms of cyanobacteria exhibit unicellular spherical and cylindrical morphologies, simple and complex multicellularity of filamentous forms, and colonial formation [8,9]. Natural morphological variations seen among cyanobacteria are shown in Figs. 1 and 2. A typical cell structure is similar to that seen in some prokaryotic cells, comprising an outer cellular cover (envelope) with a peptidoglycan wall (cell wall), resembling Gram-negative bacteria. Cell ultrastructures can be visualized using transmission electron microscopy (TEM), high-voltage electron microscopy (HVEM), or other high-resolution techniques and can exhibit considerable variability among cyanobacterial species. The genetic material or the nucleoid region comprises naked, circular DNA. Ribosomes are present, scattered throughout the cytoplasm. Cyanobacteria contain photosynthetic organelles. Photosynthesis occurs via a set of photosynthetic complexes and molecules accommodated within specialized cell membranes, called thylakoid membranes, which are freely present in the cytoplasm. Thylakoid membranes harbor the photosynthetic apparatus, i.e., photosystems, light-harvesting complexes, and phycobilisomes [10]. The fundamental physiological processes involved in cyanobacterial photosynthesis and a description of the light-harvesting complex can be found in Chapter 2. A cyanobacterial cell also contains a variety of storage inclusions that can serve as metabolic sinks during unfavorable conditions or stresses.

Fig. 1

Fig. 1 Natural morphology of unicellular cyanobacteria (A and B) and colonial formations (C–F). In panel B, a conspicuous mucilaginous sheath, resulting from secreted exopolysaccharides (EPSs), can be observed.

Fig. 2

Fig. 2 Natural morphological variations within filamentous cyanobacteria (A–K). Specialized cells, such as akinetes and heterocysts, are indicated by arrows . Abbreviations: veg , vegetative cell; ak , akinete; het , heterocyst.

2.1: Structural components

The outer parts of a cyanobacterial cell consist of a mucilaginous layer and a cell wall. The mucilaginous layer is the outermost layer covering the cell wall and protects the cell from injurious factors in the environment. In some cases, this layer is extremely conspicuous and forms a mucilaginous sheath (Fig. 1B). Cyanobacterial cells often produce mucilaginous sheaths composed of exopolysaccharides (EPSs), which range from less than 1 μm thickness to several times the filament thickness. Sheaths are often environmentally inducible and perform several putative functions, such as protection from temperature stress, desiccation, and UV radiation [11–13].

Below the mucilaginous layer is the cell wall, which comprises polysaccharides and mucopeptides. Electron microscopy has revealed that the cell wall is a relatively complex structure. The cell wall consists of two or three layers, with the inner layer lying between the outer wall layer and the plasma membrane. Cyanobacterial cell walls are similar to those of Gram-negative bacteria in that they lack the teichoic acids that are characteristic of Gram-positive bacterial walls [14]. However, the cyanobacterial peptidoglycan layer and the degree of cross-linking between the peptidoglycan chains within the murein resemble those of Gram-positive bacteria, with more cross-linking than that seen in the walls of most Gram-negative bacteria [15]. Furthermore, some cyanobacteria possess an S-layer, a crystalline, proteinaceous layer that covers the entire surface of the cell. These S-layers appear to be an important structure for filament motility [16].

The next layer is the plasma membrane, a selectively permeable membrane that encloses the cytoplasm. This structure is lipoproteinic in nature. Beneath the plasma membrane is the groundplasm, which contains various structures of different shapes and functions. In the peripheral region of the cytoplasm are thylakoid membranes, which contain photosynthetic pigments, including chlorophylls, carotenoids (such as carotenes and xanthophylls), and a pigment-protein complex known as phycobiliproteins. Several nonunit membrane inclusions or granules may be accumulated intracellularly to serve as storage bodies (see below). Some cyanobacteria also contain pseudovacuoles. Each gas vacuole comprises a number of submicroscopic units called gas vesicles. Gas vacuoles provide cyanobacteria with buoyancy, allowing them to position themselves in a place where they can take full advantage of the surrounding light and nutrient conditions; thus, gas vacuoles play a role in both light screening and buoyancy regulation.

2.2: Genetic material

The genetic material of cyanobacteria comprises naked, circular, double-stranded DNA, which usually lies coiled in a central part of the cytoplasm known as the centroplasm. This coiled DNA is equivalent to a single chromosome seen in higher organisms. The number of chromosomes (the coiled DNA) in cyanobacteria can vary. For example, in marine picocyanobacteria, such as Prochlorococcus sp., the chromosome can be monoploid or diploid [17]. The unicellular cyanobacterium Synechococcus elongatus PCC7942 is oligoploid, having between 3 and 10 copies [17]. Synechocystis sp. PCC6803 is polyploid, with a 100 copies or more [18].

As in bacteria, small circular DNA segments may also occur in cyanobacteria in addition to the nucleoid. These extrachromosomal DNA segments are known as plasmids. The existence of plasmid DNA in cyanobacteria was first observed in Anacystis nidulans [19]. The number of plasmids per cyanobacterial cell can vary from 1 to more than 10; these plasmids can also vary in size. Some representative cyanobacterial plasmids are shown in Table 1. For these cyanobacteria, complete genome sequences are available. Some cyanobacteria harbor more than 10 plasmids, such as Geminocystis sp. NIES-3709 (Table 1). The exact functions of cyanobacterial plasmids are yet to be elucidated, although it is likely that their functions include toxin production and resistance to heavy metals and antibiotics, as well as playing a role in cyanobacterial evolution [20–22].

Table 1

Data for genome size and plasmid were obtained from the KEGG database (https://www.genome.jp/kegg/).

2.3: Granules

Various storage granules occur in cyanobacteria, for carbon, nitrogen, and phosphorus storage. In addition, polyhedral bodies occur in cyanobacterial cells, which store the photosynthetic enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO). The accumulation of storage compounds in granules is one of the mechanisms that has evolved in cyanobacteria to help them adapt to environmental stress and nutrient availability. Six types of granules are discussed below.

2.3.1: Polyphosphate granules

Polyphosphate (polyP) granules, also known as volutin granules, store phosphate. They comprise orthophosphate (PO4³ −) polymers of varying lengths. Intracellular polyP granules are mainly stored in specific vacuoles called acidocalcisomes [23]. PolyP granules can bind several metals and sequester in acidocalcisomes. In cyanobacteria, polyP granules have been observed in the center of the cell and in close proximity to carboxysomes [24], in regions of the cell containing ribosomes, in close association with DNA, and in the intrathylakoid spaces [23]. PolyP granules serve as a reservoir for orthophosphate and as an energy source for fueling cellular metabolism. They also participate in maintaining adenylate and metal cation homeostasis, function as a scaffold for sequestering cations, exhibit chaperone functions, covalently bind to proteins to modify their activity, and enable normal acclimation of cells to stress conditions [23].

2.3.2: Cyanophycin

Cyanophycin (also referred to as cyanophycin granule polypeptide, CGP) is a nitrogen/carbon reserve polymer present in most cyanobacteria and nonphotosynthetic bacteria [25]. It is a nonribosomally synthesized polyamino acid consisting of aspartate and arginine. CGP accumulation occurs in most cyanobacteria under unbalanced growth conditions, including during stationary phase, light stress, or nutrient (sulfate, phosphate, or potassium) starvation. However, nitrogen starvation does not promote CGP accumulation [25,26]. Under unbalanced conditions, the amount of CGP can increase to form up to 18% of the cell dry mass [26]. CGP was formerly believed to be unique to cyanobacteria. However, following an evaluation of obligate heterotrophic bacteria genomes, it was discovered that many heterotrophic bacteria possess CGP synthetase genes [25,26].

2.3.3: Lipid droplets

Lipid droplets (LDs) (also referred to as lipid inclusions, lipid bodies, lipid globules, adiposomes, granules, oleosomes, plastoglobules, or oil bodies, depending on the field of study) act as carbon reserves and energy sources in cells [27]. LDs are composed of neutral lipids, mainly triacylglycerols (TAGs), in their core, which is surrounded by a phospholipid monolayer embedded with various integral proteins. Cyanobacterial LDs can be located throughout the cytoplasm. Small LDs (50–70 nm in diameter) have been found in the model unicellular cyanobacterium Synechocystis sp. PCC6803. Their distribution was restricted to locations among thylakoid membrane pairs and adjacent to the cytoplasmic membrane, similar to what was found in another unicellular cyanobacterium, Synechococcus sp. PCC7002, suggesting a role in thylakoid maintenance or thylakoid biogenesis [28]. In the filamentous cyanobacterium Nostoc punctiforme, the accumulation and number of LDs was found to increase upon entry into the stationary phase and following the addition of exogenous fructose, indicating a role in carbon storage. However, high levels of light stress did not increase LD numbers [29].

In nature, intracellular lipid-containing structures are found in many types of cells. These subcellular organelles are found in the cytoplasm of most eukaryotic cells, within eukaryotic plastids, and in some bacteria. In addition to their function as storage organelles, LDs are involved in other biological functions, including stress responses, hormone signaling, and plant development [30].

2.3.4: Polyhydroxyalkanoate granules

Polyhydroxyalkanoate (PHA) is a type of microbial polymer found in various microbes; it plays a role in carbon and energy storage. PHAs are a well-known family of microbial-based, biodegradable compounds. PHA accumulates when there is excess carbon availability [31,32]. After assimilation of carbon, they are processed and converted into monomer units, such as hydroxybutyrate (HB). Thereafter, monomer units are polymerized and stored in the cytoplasm. The poly-3-hydroxybutyrate (P3HB) form of polyhydroxybutyrate (PHB) is probably the most common type of PHA. Cyanobacteria can produce PHA photoautotrophically [33]. In other microbes, PHA fulfills various functions during conditions of unbalanced nutrient availability and can also protect cells against low temperatures and redox stress [32]. Although the physiological function of PHA in cyanobacteria remains unknown, this ability provides a unique opportunity to directly convert atmospheric CO2 into a biopolymer via a solar-based process. Examples of cyanobacteria capable of accumulating PHA include Nostoc muscorum NCCU-442 and Spirulina platensis NCCU-S5 [33].

2.3.5: Carbohydrate storage

Most cyanobacteria accumulate a form of soluble glycogen as their major carbohydrate store following carbon fixation by photosynthesis [34]. Photosynthetic carbon assimilation and storage of glycogen are vital processes in cyanobacterial cells during diurnal growth. These processes occur in close proximity to the thylakoid membranes, which are typically arranged around the central cytoplasmic space or project radially out from the cell poles [35]. While most cyanobacteria form soluble glycogen, some species of cyanobacteria can accumulate a different form of storage carbohydrate. This other variety of carbohydrate storage granule is referred to as cyanobacterial starch. It is considered to be a semi-amylopectin due to its lower content of longer glucan chains compared with the glucan chains found in true amylopectin. The accumulation of semi-amylopectin as a storage carbohydrate has been observed in cyanobacteria, although in a limited number of species. One of the first descriptions of semi-amylopectin accumulation in cyanobacteria was in the unicellular diazotrophic strain Cyanothece sp. strain ATCC 51142 [36]. Other carbohydrate storage granules that occur in cyanobacteria remain elusive.

2.3.6: Carboxysomes

Carboxysomes are specialized protein microcompartments that consist of a polyhedral protein shell, found in cyanobacterial cells and some autotrophic bacterial cells [37,38]. Carboxysomes are filled with RuBisCO and carbonic anhydrase to form part of the CO2-concentrating mechanism (CCM); they operate in conjunction with active uptake transporters of CO2− and bicarbonate (HCO3−), which accumulate bicarbonate in the cytoplasm of the cell. Bicarbonate is pumped into the cell, where it diffuses into the carboxysomes and is then converted to CO2 by a specific carbonic anhydrase prior to fixation. This mechanism results in the accumulation of CO2 in the vicinity of RuBisCO at a concentration high enough to promote efficient rates of carbon fixation [37].

Types of carboxysomes include α-carboxysomes, found predominantly in oceanic cyanobacteria (α-cyanobacteria), and β-carboxysomes, found mainly in freshwater cyanobacteria (β-cyanobacteria), depending on encapsulated RuBisCO and carboxysomes and their RuBisCO phylogeny [38]. As the site of CO2 fixation, it plays an essential role in the CCM of all cyanobacteria and many chemoautotrophs.

2.4: Specialized structures

Filamentous cyanobacteria are capable of cell differentiation, which is unique among prokaryotes (Fig. 2). They form specialized structures to exhibit functional cell differentiation. Here, we summarize four of the more common of these specialized structures.

2.4.1: Akinetes

Most filamentous cyanobacteria can develop perennating structures (dormant structures) in response to adverse conditions. These structures are larger than the vegetative cell, possessing thick walls, and are known as akinetes (Fig. 2). When favorable conditions return, they germinate and produce new filaments. Thus, akinetes are resting-stage cells. They contain large quantities of stored nutrients, visible as granules. Akinetes are also reproductive cells that can remain quiescent in the environment (e.g., in lake sediments, soils, etc.) during unfavorable conditions [39].

2.4.2: Hormogonia

All filamentous cyanobacteria reproduce by fragmentation of their filaments (trichomes) at more or less regular intervals to form shorter filaments, each consisting of 5–15 cells. These short filaments are referred to as hormogonia. They exhibit gliding motility and can develop into new, full-length filaments. Therefore, hormogonia represent reproductive and motile filaments.

2.4.3: Hormocysts

Some cyanobacteria produce hormocysts. These are multicellular structures that have a large, thick sheath. They may be intercalary or terminal in position and may germinate from either end or both ends to give rise to new filaments.

2.4.4: Heterocysts

Heterocysts are modified vegetative cells and the most distinctive cyanobacterial cells. They are enlarged cells with thick cell walls and are pale yellow in color (Fig. 2). Heterocysts are specialized nitrogen-fixing cells, produced by some filamentous cyanobacteria, which form during nitrogen starvation conditions. These specialized cells do not possess functioning photosynthetic apparatus because their primary function is the anaerobic fixation of atmospheric nitrogen [40]. Heterocysts fix nitrogen from dinitrogen (N2) in the air using the enzyme nitrogenase, thus providing other cells in their filament with nitrogen for biosynthesis. Nitrogenase is inactivated in the presence of oxygen, and so the heterocyst must form a microanaerobic environment. The fundamental processes involved in cyanobacterial nitrogen fixation will be described in detail in Chapter 3.

2.5: Morphology

Cyanobacteria exhibit a high degree of morphological features compared with other prokaryotes. They also present remarkable variations in morphology, from unicellular to colonial to filamentous forms (Figs. 1 and 2). Unicellular cyanobacteria include species that exhibit spherical and cylindrical morphologies. Examples include Synechococcus and Synechocystis. Cyanobacterial colonies may comprise regular or irregular distributions of cells (Fig. 1C–F). The cyanobacteria Merismopedia and Microcystis are genera that show regular and irregular distributions of cells, respectively. The members of the order Pleurocapsales possess a relatively complex colony-formation ability; their colonies can resemble filaments that exhibit branching, and their cells may be heteropolar. The number of cells can vary from 2 to several 1000 per colony [8,41].

Filamentous cyanobacteria are a diverse and morphologically complex group. Some strains are capable of cell differentiation, which is unique among prokaryotes. Filamentous cyanobacteria form chains of cells and grow as filaments [40]. Each filament consists of a sheath of mucilage and one or more cellular strands called trichomes. Filamentous forms exhibit functional cell differentiation, forming specialized structures such as heterocysts, akinetes, and hormogonia. These structures, together with the intercellular connections they possess, are considered to represent the first signs of multicellularity [39,41].

Trichome filaments can be of the homocystous type in which undifferentiated cells, for example, Oscillatoria can be found. Another type is trichome filaments that have heterocystous differentiation, i.e., they have heterocysts (Fig. 2I). An example is the genus Nostoc. Other filamentous cyanobacteria have a spiral, coiled filament, such as Spirulina (Fig. 2K). Filamentous cyanobacteria may exhibit both false and true branching. False branching is present in all orders of filamentous cyanobacteria, whereas true branching has been observed only in members of the order Nostocales. Similarly, the multiseriate growth of trichomes (a parallel succession of multiple trichomes) has only evolved in some members of the Nostocales, specifically in the order formerly referred to as the Stigonematales. On morphological grounds, cyanobacteria can be classified into five sections or taxonomic orders [8,41].

2.6: Reproduction

Most cyanobacteria reproduce via binary fission; however, some cyanobacteria have evolved interesting reproductive strategies. For instance, some unicellular cyanobacteria can produce baeocytes and exocytes, which can be differentiated from the mother cell by their size, shape, and successive multiple fission, with subsequent release into the environment [42]. Regarding unicellular ones, small and easily dispersible cells called baeocytes are formed by some strains when cell division occurs by multiple fission [41,42].

Filamentous cyanobacteria produce short, motile filaments known as hormogonia. Under unfavorable conditions, filamentous cyanobacteria, such as Nostocales, produce long-term or overwintering reproductive cells referred to as akinetes [43].

3: Ecological importance

By inhabiting all possible habitats and exhibiting a high degree of metabolic plasticity, cyanobacteria perform many crucial ecological services. For instance, they fulfill various ecological functions in the world's oceans, being important contributors to global carbon and nitrogen budgets [4]. The unique ability of some filamentous cyanobacteria to fix nitrogen has long fascinated researchers [39,40,44]. From the perspective of bioresources, cyanobacteria are valuable and prolific sources of numerous natural products with intricate chemical structures and potent biological activities. To date, more than a 1000 bioactive compounds have been described in cyanobacteria, most of which were found in filamentous cyanobacteria [45]. Bioactive compounds derived from cyanobacteria will be described elsewhere in this book.

3.1: Primary producers in food webs and use as food supplements

Among the key features of cyanobacteria in aquatic ecosystems are that they form the base of food webs and participate in symbioses with various other organisms. For example, phytoplankton consist of cyanobacteria along with algae and other groups of organisms and function as food for many species of animals. Certain species of cyanobacteria have also traditionally been used as human foodstuffs. For example, Spirulina is regularly collected for human consumption. This strain is a free-floating filamentous cyanobacterium that has been consumed as food in Africa for centuries and is now widely used as a food supplement worldwide [46–48]. Spirulina is very easily cultivated in tanks and can be used as a palatable, protein-rich food supplement for both humans and animals. The terrestrial filamentous cyanobacterium Nostoc commune has been widely used as food in Asia [49]. Another example is the freshwater unicellular cyanobacterium Aphanothece sacrum, which is consumed in Japan [50]. Thus, there is accumulating evidence to support the potential use of cyanobacteria as a foodstuff and a food supplement.

3.2: Nitrogen fixing and nutrient recycling

Several cyanobacterial species possess the ability to process atmospheric nitrogen and render it a useable organic substance. This process is known as nitrogen fixation [7,40]. The filamentous cyanobacteria possess large, specialized cells, called heterocysts, for this process. Some of the fixed nitrogen is released following the death of these cyanobacteria, resulting in the substratum becoming rich in nitrogen. For this reason, nitrogen-fixing cyanobacteria are utilized in paddy fields to provide nitrogen as a fertilizer. Filamentous cyanobacteria in the genera Anabaena and Nostoc are often used for this purpose. Nitrogen fixation is also extremely important for the growth of many types of plants. Some plants have evolved symbiotic relationships with cyanobacteria residing within their roots [51]. Cyanobacteria have also formed symbiotic relationships with many species of fungi, for example, in the case of lichens [52]. Cyanobacteria act as nitrogen fixers in various aquatic environments, making nitrogen available throughout a wide range of ecosystems [53].

Much research has been carried out to investigate the nitrogen-fixation mechanisms of cyanobacteria. However, cyanobacteria also play important roles in other nutrient cycles in a variety of habitats. They can help manage ecological niches by either directly or indirectly improving the nutrient status, including nutrients such as phosphorus, potassium, iron, and other minerals [54]. Nutrient recycling facilitates and promotes the growth of other living organisms, such as plants.

3.3: Cyanobacterial blooms and cyanotoxins

When environmental conditions suddenly shift, it can lead to dysbiosis (a reduction in microbial diversity). Upon such a shift in environmental conditions, the growth of opportunistic species can result in harmful blooms and the production of toxins. Cyanobacterial blooms can often occur in freshwater ecosystems or slow-moving water that is rich in nutrients (nitrogen and phosphorus) from sources such as fertilizers. Increasing atmospheric CO2 levels and higher water temperatures can also increase cyanobacterial growth rates, leading to blooms.

Cyanobacterial blooms have negative consequences and significant impacts on the health of aquatic species as well as animals and humans that live in or use these affected ecosystems for drinking water and/or recreational purposes. Some cyanobacterial species produce toxins [55]; so, when a bloom occurs, these toxins can accumulate in the ecosystem. Cyanobacteria, in the form of toxin-producing, deleterious phytoplankton, can potentially divert productivity into alternate food web pathways. Cyanobacterial toxins, or cyanotoxins, include potent neurotoxins, hepatotoxins, cytotoxins, and endotoxins. The production of cyanotoxins is not part of the normal growth, development, or reproduction of cyanobacteria. Therefore, they are classified as secondary metabolites [56]. Direct exposure to some cyanotoxins can result in skin rashes, while exposure to cyanobacterial neurotoxins, such as β-N-methylamino-l-alanine (BMAA), may be an environmental cause of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease [55].

4: Classical taxonomy, modern molecular phylogeny, and polyphasic approaches

Classification systems are used to describe and catalog the diversity of all living organisms. The full history of cyanobacterial classification has been described elsewhere. Currently, two different nomenclature codes are generally illustrated. These are the International Code of Nomenclature for algae, fungi, and plants and the International Code of Nomenclature of Prokaryotes in Bergey's Manual of Systematic Bacteriology [41,57].

According to the taxonomic scheme in Bergey's Manual of Systematic Bacteriology, cyanobacteria comprise five morphological orders: order I, Chroococcales (unicellular, coccoid cells that reproduce by binary fission); order II, Pleurocapsales (large cells, aggregates, or pseudofilaments that reproduce via baeocytes); order III, Oscillatoriales (simple filaments without akinetes or heterocysts); order IV, Nostocales (filamentous, divide in one plane only, heterocyst-forming); and order V, Stigonematales (filamentous, divide in one plane only, true branching, heterocyst-forming). Orders IV- and V-evolved cell differentiation, such as akinetes and heterocysts, therefore represent the most morphologically complex cyanobacteria. Examples of the representative genera of orders I–V are shown in Table 2.

Table 2

A taxonomic scheme developed on the basis of traditional methods, such as morphological, physiological, and biochemical differences, limits our understanding of the taxonomic status. These methods appear to be an insufficient tool. In particular, newly isolated species are a source of complications in taxonomic schemes. A limit of the taxonomic status according to classical taxonomy needs further clarification. Modern cyanobacterial taxonomy utilizes the ubiquitous 16S rRNA gene to construct phylogenetic relationships, which is the primary and optimal method for current taxonomic evaluation. However, paradoxes remain in both classical morphology and modern molecular phylogeny of cyanobacteria.

The taxonomic scheme for cyanobacteria can be resolved through the use of a combination of methods, i.e., collecting genotypic, chemotaxonomic, and phenotypic data to determine taxonomic positions. This combination is referred to as a polyphasic approach. Genetic information, such as 16S rRNA gene data, can be used as the primary method of evaluation. Then, several criteria, such as morphology, distinct components, and ecophysiology, can be combined and used as the secondary method. A combination of these methods is considered to provide a modern, unique, and acceptable approach for obtaining a comprehensive taxonomic scheme. However, a polyphasic approach is currently the most popular choice for the classification of microbes, including cyanobacteria [58–61].

The criteria used for a polyphasic approach to cyanobacterial classification have been reported previously [62]. The basic criteria from the phylogeny of conserved proteins, together with the arrangement of thylakoids and whether they are heterocyst-forming, were the key taxonomic features. This work was conducted in 2014; so, all suitable complete and draft genomes available at that time were used for the study. This polyphasic approach revealed eight cyanobacterial orders: Gloeobacterales, Synechococcales, Spirulinales, Chroococcales, Pleurocapsales, Oscillatoriales, Chroococcidiopsidales, and Nostocales [62,63]. This system was able to discriminate four new orders: Gloeobacterales, Synechococcales, Spirulinales, and Chroococcidiopsidales. Table 3 summarizes the classification of cyanobacteria based on the polyphasic approach used by Komárek's group [62]. Other molecular aspects and ecological analyses can also be utilized in a polyphasic approach. The use of a polyphasic approach for cyanobacterial classification can assist in the discovery of new species [58–61].

Table 3