The Pharmacological Potential of Cyanobacteria

The Pharmacological Potential of Cyanobacteria explores the bioactive compounds isolated from cyanobacteria and their relationship to human health and biotechnological applications. The book presents an overview of the chemistry and ecology of cyanobacteria, focusing on culture needs and techniques of biomass production. It is organized according to the different biological activities and biotechnological applications of compounds discovered in recent years. Besides biological activity, the mechanism of action of compounds is explained, along with molecular structure. Finally, compounds already used in therapeutics and biotechnology, as well as those in phases of approval or clinical trials are explored.

Each chapter is written by a different research group with expertise in the field and publications in peer reviewed journals. Researchers and students in pharmaceutical academic research, pharmaceutical industrial sector personnel, health professionals, and nutritionists will find this book to be very useful.

• Covers all the bioactive compounds of cyanobacteria discovered thus far
• Includes chapters by experts in the field, covering the chemistry and mechanisms of action of cyanobacteria-bioactive compounds
• Provides a general overview of organisms, from biomass production to compound isolation and evaluation of bioactivities in different cell and cell-free systems

• Language: English
• Publisher: Academic Press
• Release date: Jan 19, 2022
• ISBN: 9780128214923

Book preview

Preface

Graciliana Lopes, Marisa Silva and Vitor Vasconcelos

Cyanobacteria are among the most successful and ancient forms of life ever known. These photosynthetic autotrophs have been studied for decades as model organisms in various aspects, from photosynthesis to biotechnological applications and, more recently, for their pharmacological potential in umpteen fields. In fact, cyanobacteria are now recognized as top metabolic producers of a huge number of bioactive compounds with medical interest and that can revolutionize drug discovery and development. Allied to their metabolic capabilities, cyanobacteria benefit from a cost-effective energy-capturing ability, and high cultivation yields with minimum nutritional requirements, being extremely attractive in terms of industrial-scale production processes.

This book was designed to bring together fields in which cyanobacteria-derived compounds most stood out, with a special focus on those related to therapeutics, cosmetics, and nutrition, emphasizing unique molecules not found in higher organisms. Of the most promising compounds isolated so far, those acting as anti-inflammatories, anticarcinogens, antimicrobials, and UV protectors fill a prominent place within drug discovery programs. The metabolic richness of cyanobacteria has also been upholding their key role in the field of cosmetics and nutraceuticals, with the last occupying a prominent place in a rapidly expanding market. Apart from the pharmacological and biotechnological approach, this book does not set aside the well-known cyanobacterial toxins, warning to their substantial economic and social impacts, and drawing attention to the urgency of fully addressing algal blooms and their systematic monitoring. Additionally, and given its extreme importance, this book provides a distinctive approach to cyanobacteria systematics, by exploring general aspects and biodiversity of these organisms to discuss trends in cyanobacterial taxonomy.

Overall, The Pharmacological Potential of Cyanobacteria is intended to be a useful resource for students, researchers, and professionals working in the field of cyanobacteria, serving as a guide in the discovery, research, and application of these unique microorganisms.

Chapter 1

Trends in Cyanobacteria: a contribution to systematics and biodiversity studies

Guilherme Scotta Hentschke¹ and Watson A. Gama Junior²,    ¹CIIMAR-Interdisciplinary Centre of Marine and Environmental Research, Matosinhos, Portugal,    ²Laboratory of Phycology, Department of Biology, Federal Rural University of Pernambuco, Recife, Brazil

Abstract

Cyanobacteria emerged on Earth about 2.5 billion years ago and are the morphologically most diverse group amongst prokaryotes and the unique bacteria able to perform oxygenic photosynthesis. Most part of the cyanobacterial biodiversity is found growing in freshwater and terrestrial environments. Also, Cyanobacteria can colonize marine and extreme environments. The secondary metabolites produced by Cyanobacteria have promising bioactivities and can be applicable as pharmaceutical drugs. Currently, Cyanobacteria present 374 genera and among them, 232 genera are already confirmed by molecular tools. The current situation of Cyanobacteria systematics is complicated. Although it is mandatory to describe new genera based on the monophyletic concept of taxa, for higher taxonomical levels, all classifications systems consider para- or polyphyletic orders and subclasses. Based on that, this chapter presents the general aspects and biodiversity of Cyanobacteria and discusses trends in cyanobacterial taxonomy.

Keywords

Microbiology; Cyanobacteria; diversity; morphology; phylogeny; habitats; biotechnology; 16S rDNA

1.1 General aspects of Cyanobacteria

Cyanobacteria are the morphologically most diverse group among prokaryotes.¹,² As the unique bacteria able to perform oxygenic photosynthesis³, these organisms were understood as algae for centuries and, based on their morphology, they can be settled in three main groups: coccoid types, with unicellular or colonial shapes, the heterocytous filaments, which are able to fix nitrogen in the heterocytes, and the nonheterocytous filaments, which present no specialized cells (Fig. 1.1). Cyanobacteria can also be distinguished from other bacteria by their growth and reproductive abilities, such as filaments branching and multiple or asymmetrical cell fission. Based on these main features, for centuries Cyanobacteria were classified by taxonomists following the botanical tradition under the International Code of Botanical Nomenclature (ICBN) (formally International Code for algae, fungi, and plants), and later also under the International Code of Nomenclature of Prokaryotes (ICNP)⁴ due to their prokaryotic nature.⁵–⁷ Currently, it is recommended to describe new taxa under both codes.

Figure 1.1 Types of thalli. (A), (B) Different coccoid cyanobacteria types, showing elongated cells (Aphanothece) (A) and hemispherical cells (Chroococcus) (B); Uniseriate homocytous filament (Lyngbya) (C); Multiseriate heterocytous filament showing true branching (Stigonema) (D). Uniseriate heterocytous filaments forming fascicles (Dapisostemon) (E) and uniseriate heterocytous filament showing double false branching (Scytonema) (F). Magnification: A, D, E=200×, B=1,000×, C, F=400×.

Since the recommendation for transferring Cyanobacteria from the ICBN to the ICNP in the final 70s, these organisms started to be more intensively studied by polyphasic taxonomy, which characterizes them according to ecological, morphological and biochemical features, but mainly by using molecular tools. Based on 16S rDNA, and more recently genomic data, it is known that morphology does not reflect the true evolutionary relations among Cyanobacteria taxa.⁸ In fact, coccoid and nonheterocytous filaments are close related, and currently it is known that the multicellularity in Cyanobacteria evolved independently, appearing at least four times along the evolution of this group.⁹,¹⁰ On the other hand, it was also confirmed that heterocytous filaments are monophyletic in the Nostocales order, and their clade is close related to the Chroococcidiopsis-like Cyanobacteria, a coccoid type.⁸

Nevertheless, even morphology is proved to not reflect the whole phylogenetic scenario in Cyanobacteria, it is still important. In practice, water quality control in reservoirs, lakes, and rivers is still performed first by morphological identification of taxa, in many places over the world.¹¹ Also, morphology supports many studies regarding Cyanobacteria distribution, ecology, and physiology. For systematics, morphological studies are the link between the classic information and the sequences datasets used for the polyphasic approach analyses. Much of the known biodiversity of Cyanobacteria is still based only on classic (or morphological) taxonomy. This knowledge is important to set reference sequences for morphogenera and confirm them by phylogenetic studies. The molecular tools are very efficient to reflect the phylogenetic relationships among taxa and are also very sensitive in the detection of biodiversity. However, they require sequence libraries for taxa identification, which are retrieved from isolated strains previously identified by morphological analysis. Consequently, the correct morphological identification of taxa must be properly done, otherwise it will lead to misidentifications—which is a significant issue in cyanobacterial sequence libraries,¹²,¹³ especially to simple forms.¹⁴,¹⁵ Besides, some morphological markers are good synapomorphies to some groups, as the presence/absence of heterocytes (Nostocales), presence/absence of cross-walls (Spirulinales), and nanocytes/baeocytes formation (Pleurocapsales/Chroococcidiopsidales).¹⁶–¹⁸ More recently, a new order was proposed in Cyanobacteria, Gloeomargaritales, to accommodate Gloeomargarita lithophora, a very particular coccoid type, which has many carbonate granules inside its cytoplasm and is very distinct from other Cyanobacteria based on a multigene analysis.¹⁹ Found growing on calcareous rocks, this unicellular cyanobacterium is close related to the ancestor which has ended up in chloroplasts through a singular Endosymbiotic event.²⁰ Regarding ancestry and evolution of Cyanobacteria, there are also new proposals to accommodate the Melainbacteria as a class inside Cyanobacteria Phylum, since they are very close phylogenetically related to each other.²¹ These uncultured bacteria were first found in human gut and groundwater samples and are nonphotosynthetic prokaryotes, which suggest that Cyanobacteria first evolved from a nonphotosynthetic and fermentative ancestor.²²

All this diverse and complex phylogenetic history has been strongly related to the ancient existence of Cyanobacteria, dating from 2.5 billion years ago.²³,²⁴ This partially explains why Cyanobacteria can be found almost at any place on the planet, growing in water (marine and freshwater), on land habitats (as rocks, topsoil, tree barks), and in extreme environments, like hot springs, alkaline and salty waters, deserts and arid regions (forming the biocrusts) and even inside caves, where the light rates are very low.³,²⁵,²⁶ To grow in this wide range of environments, and to resist multiple stress conditions,²⁷ Cyanobacteria developed many survival adaptations, each one specific to their habitat. As an example of these skills, it is possible to cite the variety of chlorophylls produced in the cyanobacterial group, as a, b, and the exclusive d and f types. The ability of Cyanobacteria to fix nitrogen is also unique among the oxy-phototrophic organisms, and it is very worth to plants that have Cyanobacteria as their symbionts, like Azolla, Anthoceros, and Cycas.²⁸ Cyanobacteria are also found in symbiotic relationships with fungi, forming the Cyanolichens.²⁹ As seen, the ecological role of Cyanobacteria is vast and has gained visibility especially in the primary productivity of the Oceans,³⁰ and on the biocrusts of arid regions.³¹,³²

Furthermore, Cyanobacteria have also gained space in biotechnological studies and applications. With the commercial product Spirulina (which is actually an Arthrospira or Limnospira species—see Nowicka-Krawczyk et al.³³), Cyanobacteria are already present in food and cosmetic products.³⁴,³⁵ Moreover, the ability to survive over environmental stress is mirrored in the production of secondary metabolites, many of them, exclusively produced by Cyanobacteria, with promisor bioactivities that can be applicable as pharmaceutical drugs, as antifungal, antioxidant, anticancer, bactericide, antiviral, antiobesity and in psoriasis treatment.³⁶–⁴¹ Besides, extracts have also activities with economic importance, such as antifouling,⁴² and have been applied in medical tests to help in the treatment of chronic wounds, showing effective results.⁴³ Cyanobacteria can be used as biofertilizers and increase yield in multiple cultures. Beyond that, as technology advances, new demands for humanity arise. Recently, two cyanobacterial strains were found to be able to survive outside the International Space Station (ISS), resisting extreme cold and radiation. In this way, Cyanobacteria are strong candidates to help human beings to colonize other planets.⁴⁴

However, it is also important to highlight the power of Cyanobacteria in forming harmful blooms in aquatic ecosystem, which is a response to the eutrophication of waters and has impacts on public health and economy, especially in areas with warm/hot and dry weather and shallow waters. The capacity of producing toxins and being protagonists in Harmful Algal Blooms (cyanobacterial Harmful Algal Blooms—CyanoHABs) have put Cyanobacteria in evidence, and many efforts have been made to understand and control this phenomenon.⁴⁵

1.2 Cyanobacteria biodiversity

Currently, Cyanobacteria present 1,468 valid described species (CyanoDB—⁴⁶), 374 genera, 56 families, and eight orders. Among them, 232 genera are already confirmed by molecular tools and have their phylogenetic position confirmed by the 16S rDNA. The Nostocales is the most diverse order with 84 genera confirmed by 16S rDNA and 45 morphogenera, followed by Synechococcales and Oscillatoriales. The number of molecular genera is bigger than morphogenera in all of these orders, but for Chroococcales and Pleurocapsales, the lack of molecular studies is evidenced by the fact that they present more morphogenera than molecular genera (Fig. 1.2). Considering that, molecular studies on Chroococcales and Pleurocapsales would be very useful to Cyanobacteria systematics and to increase the knowledge about their biodiversity, improving phylogenies and taxa classification, in families and orders levels. Moreover, even for orders which are better studied, as Nostocales, Synechococcales, and Oscillatoriales, there are still 95 morphogenera that must be confirmed by molecular studies.

Figure 1.2 Relation between the number of existing morphogenera and genera confirmed by 16S rDNA phylogenies in cyanobacterial orders.

In fact, the molecular studies have been responsible for the great increase in the number of taxa described since 2010. Comparing the number of new genera descriptions over the centuries, they started in the 19th, but we are in the second decade of the 21st century, and 154 new genera were described during these 20 years, representing 46% of Cyanobacteria total known biodiversity. This boom of new genera overlaps the establishment of polyphasic approach in the Cyanobacteria taxonomy and the wider access to molecular sets, which proved many taxa in Cyanobacteria to be polyphyletic, and lead them to be described as new. Before that, the number of genera descriptions was also remarkable during the 1980s, due to the publications of Prof. Komárek’s books and papers. According to Nabout et al.,⁴⁷ this author and Prof. Anagnostidis were responsible for the description of 834 species, 30% of the total diversity to that date. Another peak of taxonomical descriptions is shown during the 1880s, when Cyanobacteria starting points were established (Fig. 1.3).

Figure 1.3 Number of new cyanobacterial genera described through time.

Considering habitats, commonly cyanobacterial genera can colonize more than one habitat. Most part of this biodiversity is found growing in freshwater (197 genera) and terrestrial (162 genera) environments. On the other hand, even marine Cyanobacteria are very diverse (106 genera), this is proportionally the group with fewer molecular studies (Fig. 1.4).

Figure 1.4 Relative proportion (%) of cyanobacterial morphogenera and genera confirmed by 16S rDNA phylogenies according to their habitats.

Consequently, the biodiversity for this habitat has great potential to be substantially increased in the future, since studies on marine Cyanobacteria are gaining force and importance in the last years. This recent attention in marine Cyanobacteria is due mainly to the great capacity of these types to produce secondary metabolites with biological activities.⁴⁰ Extreme environments are poorly studied as well and, currently, only 39 genera are known to grow in these habitats. The relation between order and habitats shows that Nostocales is predominantly terrestrial (78 of 129 genera), while Synechococcales (66 of 103 genera) and Oscillatoriales (41 of 65 genera) are predominantly found growing in freshwater environments (Fig. 1.5).

Figure 1.5 Relative proportion (%) of described genera according to their habitats and respective cyanobacterial order.

In order to study this great Cyanobacteria diversity, it is important to culture them. This has stimulated the isolation of cyanobacterial diversity in culture collections all around the World. Types and reference strains are mainly found in Pasteur Culture Collection (PCC, France), Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC, Portugal), Culture Collection of Autotrophic Organisms (CCALA, Czech Republic), Coimbra Collection of Algae (ACOI, Portugal), Culture Collection of Algae at The University of Texas at Austin (UTEX, United States), NIES Microbial Culture Collection (Japan), Centro de Energia Nuclear na Agricultura (CENA, Brazil), Coleção de Culturas de Algas, Cianobactérias e Fungos do Instituto de Botânica (CCIBt, Brazil). Besides being the repository of cyanobacterial biodiversity, the strains kept in these collections can be used in bioprospection studies, which has revealed a promising potential in the biotechnological field.⁴⁸–⁵⁰

1.3 Classification systems and future challenges for Cyanobacteria taxonomy

Classification systems are created by humans on their desire to organize space and put names on everything surrounding them. Although this is essential for the development of biological sciences, it is almost impossible to precisely recreate the evolutionary relations among organisms due to many factors. For higher plants, for example, interbreeding species and spatial and temporal variations in populations complicate the definition and delimitation of species and higher taxonomical levels.⁵¹,⁵² For Prokaryotes, the haploid condition, horizontal gene transfer, higher cell multiplication rates, higher sensibility to environmental changes, associated with the higher probability that a favorable mutation may persist within a population, rather than being lost by chance⁵³,⁵⁴ and the simple morphology turns the delimitation of species even more difficult. These problems are also valid for higher taxonomical levels, reflecting, in the case of Cyanobacteria, in the polyphyletic orders which are currently in use. Another problem in the taxonomy of Cyanobacteria is that the descriptions of new taxa under both botanical and bacteriological codes are based on cultures originated from one or a few individuals. These individuals represent species, genera, or even families with unknown genetic variability. This lack of knowledge in the genetic variability of cyanobacterial species also contributes to making the delimitation of species in Cyanobacteria not clear. Currently, although formally species should be considered as groups with same genotype and morphotype with stable phenotypic features, and more or less stable and distinct ecological limits,⁵⁵ there is no consensus about which genes should be used to separate species or what is the genotypic variance that should be considered in species-level taxonomy. Consequently, authors judge and separate species mainly according to morphological and ecological characters. The secondary structures of the 16S-23S rDNA intergenic spacer are also frequently subjectively used, and no statistics or phylogenetic analysis is applied in these cases.

Although there is no full definition of species criteria, at genus level, the 16S rDNA is mandatory to describe new genera based on the monophyletic concept of taxa.⁵⁶ This gene is conserved and presents ~1,500 base pairs, being suitable for phylogenetic analysis and very concise at genus level. Even considering that the 16S rDNA is reliable to reconstruct evolutionary relations among genera, some problems in cyanobacterial 16S phylogenies arise: (1) there are many misidentified strains in public genomic databases; (2) many reference strains and type species present short 16S sequences (less than 1,200 base pairs); (3) 16S rDNA of type specimens described before the polyphasic taxonomy Era are hardly accessed (rare exceptions are registered, but with short sequences, see Palinska et al.⁵⁷). Among these problems, the third one is the most problematic. Assign neotypes or reference strains for old morphotypes is very complicated, because most of them are poorly morphologically and ecologically described, and sometimes cannot be related to the specimens sampled today. Beyond that, even with precise descriptions, it is not possible to know if this old morphotype can represent one or more species if we consider the possibility of cryptogenera or cryptic species. Consequently, these problems complicate also the classification of Cyanobacteria in higher taxonomic levels, and although we use the monophyletic genera concept, at higher levels, families and orders are still polyphyletic.

Trying to solve that, with the evolution of technology and molecular studies, taxonomists started to use concatenated proteins and whole-genome sequences to perform cyanobacterial phylogenies, but, surprisingly, the results are very similar to those with 16S rDNA.⁵⁸,⁵⁹ The current trees show polyphyletic orders and families and some factors must be highlighted in these cases: (1) the evolutionary history of many genes are intermixed; these genes have different mutation rates and are susceptible to different horizontal transfer events in time and space; (2) horizontal transfer of genes from one individual to other individuals with distant evolutionary histories have effects in phylogenies, and because of that, these taxa can eventually turn out to be phylogenetically close related, even with distant evolutionary history. The consequence of these two factors is that different concatenated genes with different lengths, different mutation rates, and different horizontal transfer frequency can have different influence in phylogenies. Longer and more variable genes have more influence in phylogenies than short and conserved genes, which is not suitable for higher taxonomical levels studies. Another problem to be considered in whole-genome phylogenies is that, currently, a few genera present these data. Beyond that, phylogenies are influenced by alignments and phylogenetic models changing.

Regarding the classification systems based on polyphasic trend to Cyanobacteria, the information about Cyanobacteria higher taxa has mostly been reaffirmed throughout the years. The classification system of Hoffmann et al.⁶⁰ was the first in Cyanobacteria to consider genetics, ultrastructure, and morphology to separate the taxa. This system was also the first to put coccoid and filamentous genera together at the same classification level. The polyphasic approach used in this system related ultrastructural features (thylakoids arrangement) with the phylogenetic results to separate the paraphyletic subclasses Synechoccophycideae and Oscillatoriophycideae. The only monophyletic lineage presented was Nostocophycideae. This classification system accepted polyphyletic orders and later in 2019, the importance of the thylakoid arrangement to separate cyanobacterial lineages was refuted.⁶¹ Currently, cyanobacterial taxonomists use the classification system proposed by Komárek et al.,⁵⁹ which presents a robust phylogenetic analysis considering 31 conserved protein sequences and emphasizes that genera must be monophyletic, although it considers para- and polyphyletic orders. The problem with polyphyletic orders in Cyanobacteria is confirmed by Chen et al.,⁵⁸ who built a phylogenetic tree with genome sequences of Cyanobacteria. Interestingly, the results are very similar to our Maximum Likelihood phylogeny (Fig. 1.6), which were obtained with 330 16S rDNA sequences (857 positions analyzed) of type species and published reference strains of Cyanobacteria (Table 1.1).

Figure 1.6 16S rDNA Maximum likelihood phylogeny of 330 cyanobacterial reference strains. The bootstrap number was 1000 and there were a total of 877 positions analyzed.

Table 1.1