Nitrogenase Enzyme Research Articles

Reconstruction of Nitrogenase Predecessors Suggests Origin from Maturase-Like Proteins.

The evolution of biological nitrogen fixation, uniquely catalyzed by nitrogenase enzymes, has been one of the most consequential biogeochemical innovations over life’s history. Though understanding the early evolution of nitrogen fixation has been a longstanding goal from molecular, biogeochemical, and planetary perspectives, its origins remain enigmatic. In this study, we reconstructed the evolutionary histories of nitrogenases, as well as homologous maturase proteins that participate in the assembly of the nitrogenase active-site cofactor but are not able to fix nitrogen. We combined phylogenetic and ancestral sequence inference with an analysis of predicted functionally divergent sites between nitrogenases and maturases to infer the nitrogen-fixing capabilities of their shared ancestors. Our results provide phylogenetic constraints to the emergence of nitrogen fixation and are consistent with a model wherein nitrogenases emerged from maturase-like predecessors. Though the precise functional role of such a predecessor protein remains speculative, our results highlight evolutionary contingency as a significant factor shaping the evolution of a biogeochemically essential enzyme.

NIFtHool: an informatics program for identification of NifH proteins using deep neural networks.

Atmospheric nitrogen fixation carried out by microorganisms has environmental and industrial importance, related to the increase of soil fertility and productivity. The present work proposes the development of a new high precision system that allows the recognition of amino acid sequences of the nitrogenase enzyme (NifH) as a promising way to improve the identification of diazotrophic bacteria. For this purpose, a database obtained from UniProt built a processed dataset formed by a set of 4911 and 4782 amino acid sequences of the NifH and non-NifH proteins respectively. Subsequently, the feature extraction was developed using two methodologies: (i) k-mers counting and (ii) embedding layers to obtain numerical vectors of the amino acid chains. Afterward, for the embedding layer, the data was crossed by an external trainable convolutional layer, which received a uniform matrix and applied convolution using filters to obtain the feature maps of the model. Finally, a deep neural network was used as the primary model to classify the amino acid sequences as NifH protein or not. Performance evaluation experiments were carried out, and the results revealed an accuracy of 96.4%, a sensitivity of 95.2%, and a specificity of 96.7%. Therefore, an amino acid sequence-based feature extraction method that uses a neural network to detect N-fixing organisms is proposed and implemented. NIFtHool is available from: https://nifthool.anvil.app/.

NifH gene analysis of endophytic bacteria of sweet potato under various climatic locations

Beyond the nodule symbiosis process, some endophytic bacteria can convert the atmospheric nitrogen to available form for their own and the inhabiting host plants. These bacteria possess a nitrogenase enzyme complex involved in producing ammonia from atmospheric dinitrogen. The nitrogenase reductase subunit of the enzyme complex is governed by the nifH gene and is being used as a suitable marker. In the present study, the nifH gene containing endophytic bacteria had been identified from two sweet potato cultivars grown in different locations. Both the cultureindependent and culture-dependent methods were used to identify the nitrogen-fixing bacteria in sweet potatoes. The nifH sequences closest to Azospirillum, Bradyrhizobium, Herbaspirillum, Klebsiella, Kluyvera, Paenibacillus, Pseudacidovorax, Rhizobium and Sphingomonas were identified in this study. It was supposed that soils and varieties have more effects than climatic factors on nitrogen-fixing bacteria of sweet potato. These nitrogen-fixing bacteria could be utilized to develop biofertilizers helpful for sustainable agriculture.

A biochemical, physiological and molecular evaluation of how the herbicide 2, 4-dichlorophenoxyacetic acid intercedes photosynthesis and diazotrophy in the cyanobacterium Nostoc muscorum Meg 1.

Among the non-target microorganisms residing in crop fields that are potentially vulnerable to herbicides are cyanobacteria. They contribute to the maintenance of soil quality and fertility and hence are considered to be an important component of soil microflora. Consequently, the present study was aimed to check the influence of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on some major parameters of carbon (CO2) and nitrogen (N2) fixations of a cyanobacterium Nostoc muscorum Meg 1 isolated from a rice field in Cherrapunji, Meghalaya, India. These include various photosynthetic pigments, the oxygen-evolving complex activity of the PSII, the protein contents of RuBisCO, D1 protein, isocitrate dehydrogenase (IDH), nitrogenase and glutamine synthetase (GS) enzymes, the heterocyst percentage, nitrogenase and GS enzyme activities, and production of total proteins and carbohydrates in the cyanobacterium in a varying range of 50 to 125 ppm doses of 2,4-D. The mRNA levels of several proteins were also analyzed. Besides carotenoid concentration that enhanced at 50 ppm, all other parameters were compromised by 2,4-D in a dose-dependent manner resulting in a reduction in photosynthetic and N2-fixing activities. The negative effect on N2-fixation was partly due to compromised IDH activity. RT-PCR analysis further showed that these negative effects were initiated at transcription levels as mRNA contents of all enzymes studied were found compromised under 2,4-D exposure. The scanning and transmission electron microscopy further revealed herbicide induced adverse changes in the morphology and ultrastructure of the organism. The significance of the work lies in its detailed analysis of the effect of 2,4-D at biochemical, physiological, and molecular levels.

Dinitrogen Binding Relevant to FeMoco of Nitrogenase: Clear Visualization of σ‐Donation and π‐Backdonation from Deformation Electron Densities around Carbon/Silicon‐Iron Site

AbstractDinitrogen (N2) binding and electron transfer reduction of N2 to ammonia (NH3) by the FeMoco cofactor of the nitrogenase enzyme are captivating. They are a part of the textbook for general chemistry. The nature of N2 bonding by reduced FeMoco is speculated based on the experimental evidence. The inorganic core MoFe7S9C1− possesses a Fe6(μ6‐C4−) unit. The mode of N2‐binding at one of the Fe‐centers of the elusive Fe6(μ6‐C4−) unit and the role of light element C4− is intriguing. In the past, the mode of N2‐binding and the kinetics of N2 reduction have been studied by spectroscopic and other tools. Herein, we report on the energy decomposition analysis coupled with natural orbital for chemical valence (EDA‐NOCV) calculations/analyses to shed light on the deeper insight of the N2 binding and especially on the influence of the C‐atom of previously reported Fe‐complexes with an EP3 donor set (E=C, Si). The role of the C‐atom in the iron‐carbon site has been studied by elaboration with deformation electron densities. The intrinsic interaction energy of the bond between Fe and N2 and pairwise orbital interactions between them have been quantitatively estimated. The influence of σ‐donation of three phosphine ligands and their effects on the Fe−N2 bond have been thoroughly studied.

Dynamic diel proteome and daytime nitrogenase activity supports buoyancy in the cyanobacterium Trichodesmium.

Cyanobacteria of the genus Trichodesmium provide about 80 Tg of fixed nitrogen to the surface ocean per year and contribute to marine biogeochemistry, including the sequestration of carbon dioxide. Trichodesmium fixes nitrogen in the daylight, despite the incompatibility of the nitrogenase enzyme with oxygen produced during photosynthesis. While the mechanisms protecting nitrogenase remain unclear, all proposed strategies require considerable resource investment. Here we identify a crucial benefit of daytime nitrogen fixation in Trichodesmium spp. that may counteract these costs. We analysed diel proteomes of cultured and field populations of Trichodesmium in comparison with the marine diazotroph Crocosphaera watsonii WH8501, which fixes nitrogen at night. Trichodesmium's proteome is extraordinarily dynamic and demonstrates simultaneous photosynthesis and nitrogen fixation, resulting in balanced particulate organic carbon and particulate organic nitrogen production. Unlike Crocosphaera, which produces large quantities of glycogen as an energy store for nitrogenase, proteomic evidence is consistent with the idea that Trichodesmium reduces the need to produce glycogen by supplying energy directly to nitrogenase via soluble ferredoxin charged by the photosynthesis protein PsaC. This minimizes ballast associated with glycogen, reducing cell density and decreasing sinking velocity, thus supporting Trichodesmium's niche as a buoyant, high-light-adapted colony forming cyanobacterium. To occupy its niche of simultaneous nitrogen fixation and photosynthesis, Trichodesmium appears to be a conspicuous consumer of iron, and has therefore developed unique iron-acquisition strategies, including the use of iron-rich dust. Particle capture by buoyant Trichodesmium colonies may increase the residence time and degradation of mineral iron in the euphotic zone. These findings describe how cellular biochemistry defines and reinforces the ecological and biogeochemical function of these keystone marine diazotrophs.

The Three Sisters of Agriculture: An Active Learning Activity on Symbiotic Nitrogen Fixation

This active learning exercise introduces students to the plant microbiome and the contributions that bacteria make to plant growth and food production. The Three Sisters are an ancient indigenous practice in which three plant crops and the associated microbiome contribute to each other&rsquo;s growth. In this symbiotic relationship, the associated bacteria (<em>Rhizobium leguminosarum</em> biovar <em>phaseoli</em>) inhabit root nodules in bean plants, converting atmospheric nitrogen to ammonia through the process of nitrogen fixation. The plant takes up the ammonia as its nitrogen source, and provides the bacteria with organic carbon. The Lesson contains a pre-class reading, a 50-minute class session, and an after-class reading. In the first round of small group work during the class, students discuss and report back on the symbiosis between the three plants and between the bacteria and the plants, and the contributions to sustainable agriculture. In the second round of discussion and reporting, students discuss nitrogen fixation, emphasizing the <em>nod</em> genes for polypeptides involved in forming the root nodules and the <em>nif</em> genes that encode the nitrogenase enzyme complex that carries out nitrogen fixation. The after class reading provides students with an example of enhancing plant growth by adding nitrogen-fixing bacteria externally to beans. Altogether, this exercise provides students with a real life scenario relevant to sustainable agriculture. <em>Primary Image:</em>&nbsp;Nitrogen fixation in bean root nodules. Nodule formation starts with the secretion of flavonoids from bean roots, leading to the induction of nodulation factors in the bacteria. After root curling and nodule formation, the enzyme nitrogenase catalyzes the reaction from atmospheric nitrogen to plant accessible ammonia.

Calculating the chemical mechanism of nitrogenase: new working hypotheses.

The enzyme nitrogenase converts N2 to NH3 with stoichiometry N2 + 8H+ + 8e- → 2NH3 + H2. The mechanism is chemically complex with multiple steps that must be consistent with much accumulated experimental information, including exchange of H2 and N2 and the N2-dependent hydrogenation of D2 to HD. Previous investigations have developed a collection of working hypotheses that guide ongoing density functional investigations of mechanistic steps and sequences. These include (i) hypotheses about the serial provision of protons and their conversion to H atoms bonded to S and Fe atoms of the FeMo-co catalytic site, (ii) the migration of H atoms over the surface of FeMo-co, (iii) the roles of His195, (iv) identification of three protein channels, one for the ingress of N2, a separate pathway for the passage of exogenous H2 (D2) and product H2 (HD), and a hydrophilic pathway for egress of product NH3. Two additional working hypotheses are described in this paper. N2 passing along the N2 channel approaches and binds end-on to the exo coordination position of Fe2, with favourable energetics when FeMo-co is pre-hydrogenated. This exo-Fe2-N2 is apparently not reduced but has a promotional role by expanding the reaction zone. A second N2 can enter via the N2 ingress channel and bind at the endo-Fe6 position, where it is surrounded by H atom donors suitable for the N2 → NH3 conversion. It is proposed that this endo-Fe6 position is also the binding site for H2 (generated or exogenous), accounting for the competitive inhibition of N2 reduction by H2. The HD reaction occurs at the endo-Fe6 site, promoted by N2 at the exo-Fe2 site. The second hypothesis concerns the most stable electronic states of FeMo-co with ligands bound at Fe2 and Fe6, and provides a protocol for management of electronic states in mechanism calculations.

Nitrogen Fixing Bacteria and Their Application for Heavy Metal Removal: A Mini Review

Nitrogen is a critical component of biological systems and typically serves as a constraint on production in both aquatic and terrestrial environments, although its shortage has been compensated for through the process of biological nitrogen fixation. Nitrogen fixation is a critical microbial activity that utilises nitrogenase enzymes to convert dinitrogen (N2) gas to ammonia (NH3). It is carried out by a diverse spectrum of bacteria known as nitrogen fixing bacteria. These include free-living bacteria such as Azotobacter, Bacillus, Beijerickia, and Clostridium, associative bacteria such as Azospirillum, Enterobacter, and Pseudomonas, and bacteria that form symbiotic associations with legumes such as Rhizobium and actinorrhizal plants such as Frankia. These bacteria contribute significantly to plant growth by producing phytohormones (such as auxins, cytokinins, gibberelins, and indole acetic acid), reducing the incidence of plant diseases through the production of siderophores and cell wall degrading enzymes, and increasing phosphorus nutrition via phosphate solubilization. Additionally, they remove heavy metal ions from solutions through a process called biosorption, which is a feasible, natural, environmentally benign, and economically viable technique of remediating heavy metal-contaminated environments.

DETECTION OF NITROGENASE PRODUCING BACTERIA FROM THE SOIL OF LIWA BOTANICAL GARDEN

Liwa Botanical Gardens is an ex-situ conservation area for various types of plants. Each plant produces organic matter that will provide nutrients for the growth of nitrogen-fixing bacteria. This indicates the existence of an environment that supports the growth of nitrogen-fixing bacteria. Nitrogen is one of the nutrients needed by plants for their growth. However, the abundance of nitrogen in the atmosphere cannot be utilized directly by plants but needs to transform into ammonium and nitrate first. This transformation can be done by nitrogen-fixing bacteria through an enzymatic process. This research aims to obtain bacterial isolates that can fix nitrogen. Nitrogen-fixing bacteria were isolated using Nutrient Agar (NA) medium and furthered by nitrogenase activity detection test with semi-solid Nitrogen Free Bromothymol Blue (NFB). Nitrogen-fixing bacteria are characterized by color changes in the medium. The results obtained 22 isolates with 3 isolates detected capable of producing nitrogenase enzymes, namely TBP B3, TB1 B2, and TMA2 B2.

ВЫЯВЛЕНИЕ ГЕНОВ ФЕРМЕНТОВ У БАКТЕРИЙ ВИДА BACILLUS SUBTILIS МЕТОДОМ REAL-TIME PCR

Soil is a vital and valuable natural resource that sustains life on earth. Appropriate soil functioning depends on the balance of its structure and composition, as well as physical, chemical and biological properties. Often, this balance is disturbed under the influence of various abiotic, biotic and anthropogenic factors. Therefore, soil restoration is of great importance in order to prevent possible adverse effects on living systems and to preserve the environment for future generations. Various studies confirmed the effectiveness of introduction of rhizobacteria to improve soil fertility, increase growth and productivity of agricultural crops. Bacillus subtilis is one of the most common rhizobacteria used in agriculture. Many B. subtilis strains are capable of fixing atmospheric nitrogen, solubilizing phosphorus and potassium, thereby contributing to an increase in the amount of macroelements necessary for plant nutrition in soil. The aim of this work was to search for genes responsible for synthesis of phytase, nitrogenase, and alkaline phosphatase enzymes in strains of bacteria of Bacillus subtilis species by real-time PCR. To determine the presence of genes encoding the synthesis of the desired enzymes in Bacillus subtilis, an in-silico analysis of the annotated genomes of this bacterial species presented in the NCBI information database was carried out. Then the selection of primers for screening the target spots was made. According to the results of the study, 10 out of 19 isolated Bacillus subtilis strains contained all three required DNA regions responsible for synthesis of phytase, nitrogenase, and alkaline phosphatase enzymes.

Insights into Tuning of Mo‐Based Structures toward Enhanced Electrocatalytic Performance of Nitrogen‐to‐Ammonia Conversion

Ammonia is commonly produced via the Haber−Bosch process, which is energy intensive with emission of substantial greenhouse gases. As one alternative method, electrochemical nitrogen reduction reaction (NRR) can be driven by the renewable energy and realized at ambient conditions for sustainable NH3 synthesis. However, the reaction suffers from high overpotentials and low selectivity due to the potential overlap with competing proton reduction. As inspired by the natural nitrogenase enzymes, Mo sites are particularly investigated to exhibit the potential for catalyzing NRR. Herein, the recent progresses in modifying Mo‐based catalysts toward enhanced NRR performance are discussed. The modification strategies vary from tuning of metal cores, coordination spheres, and assembled structures. The modification strategies can both efficiently tune the chemisorption energy and geometry of N2, as well as hydrogenated N derivatives on the Mo sites.

Computational evaluation of FeMo heteroatom coeffect induced high electroreduction activity of N2-to-NH3

Fe-Mo system are recognized as the most successful nitrogenases system towards the synthesis of ammonia. At the same time, Fe or Mo plays a key role in nitrogenase enzymes together with the most common catalyst for nitrogen reduction reaction (NRR). Herein, we designed and explored NRR catalysts of bimetallic Fe-Mo atoms incorporating on three 2D materials, including graphdiyne (GD), Nitrogen atoms substituted graphene (G-N6) and C2N, by means of density functional theory. Fe-Mo atoms supported on C2N is calculated to the most promising one owing to its lowest limiting potential (−0.17 V) and excellent selectivity against hydrogen evolution reaction. The superior performance of FeMo dual-atom NRR catalysts has also been verified by comparing to individual Fe or Mo involved catalysts on account of synergistic effects. Besides, the high NRR activity of FeMo@C2N could be ascribed to the high localized electronic states of 3d/4d and 2p orbitals near the Fermi level, providing the Fe-Mo active sites with optimal electronic state. Our studies would further inspire some experimental and theoretical researchers to find out the efficient catalysts with high activity for the synthesis of ammonia at room temperature.

Structural analysis of the reductase component AnfH of iron-only nitrogenase from Azotobacter vinelandii

Biological nitrogen fixation, the conversion of atmospheric dinitrogen into bioavailable ammonium, is exclusively catalyzed by the enzyme nitrogenase that is present in nitrogen-fixing organisms, the diazotrophs. So far, three different nitrogenase variants, encoded in their corresponding, distinct gene clusters, have been found in nature. Each one of these consists of a catalytic dinitrogenase component and a unique, ATP-dependent reductase, the Fe protein. The three variant nitrogenases differ in the composition of the active site and contain either molybdenum, vanadium or only iron in the dinitrogenase component. Here we present the 2.0 Å resolution crystal structure of the ADP-bound reductase component AnfH of the iron-only nitrogenase from the model diazotroph Azotobacter vinelandii. A comparison of this structure with the ones reported for the two other Fe protein homologs NifH and VnfH in the ADP-bound state shows that all are adopting the same conformation. However, cross-reactivity assays with the three nitrogenase homologs revealed AnfH to be compatible with iron-only nitrogenase and to a lesser degree with the vanadium-containing enzyme, but not with molybdenum nitrogenase.

Biotechnological approaches to develop nitrogen-fixing cereals: A review

ricultural yields are often limited by nitrogen (N) availability, especially in countries of the developing world, whereas in industrialized nations the application of chemical N fertilizers has reached unsustainable levels that have resulted in severe environmental consequences. Finding alternatives to inorganic fertilizers is critical for sustainable and secure food production. Although gaseous nitrogen (N2) is abundant in the atmosphere, it cannot be assimilated by most living organisms. Only a selected group of microorganisms termed diazotrophs, have evolved the ability to reduce N2 to generate NH3 in a process known as biological nitrogen fixation (BNF) catalysed by nitrogenase, an oxygen-sensitive enzyme complex. This ability presents an opportunity to improve the nutrition of crop plants, through the introduction into cereal crops of either the N fixing bacteria or the nitrogenase enzyme responsible for N fixation. This review explores three potential approaches to obtain N-fixing cereals: (a) engineering the nitrogenase enzyme to function in plant cells; (b) engineering the legume symbiosis into cereals; and (c) engineering cereals with the capability to associate with N-fixing bacteria.

Cover Feature: Tailoring Quantum Dot Sizes for Optimal Photoinduced Catalytic Activation of Nitrogenase (ChemSusChem 24/2021)

The Cover Feature shows how nanomaterial sizes affect the light-induced electron transfer process with the nitrogenase enzyme. When 2.3 nm CdSe quantum dots are utilized for the photocatalytic hydrogen generation process, an 8-fold increase can be gained as compared with larger-sized quantum dots. Additionally, cysteine-mutated nitrogenase at the electron acceptor side (P-cluster) does not lead to enhanced catalytic performance. More information can be found in the Communication by M. M. Meirovich et al.

Tailoring Quantum Dot Sizes for Optimal Photoinduced Catalytic Activation of Nitrogenase.

Many efforts have been directed towards elucidating the nitrogenase structure, its biocatalytic activity, and methods to artificially activate it by external stimuli. Here, we investigated how semiconductor nanoparticles (NPs) with sizes ranging between 2.3-3.5 nm form nano-biohybrids with the nitrogenase enzyme and enable its photoinduced biocatalytic activity. We examined two homogenously synthesized quantum dots (QDs), CdS, CdSe, and two nitrogenase variants, the wild-type and a cysteine-mutated. We show that the cysteine-mutated variant does not enhance the hydrogen generation amounts, as compared with the wild type. Nevertheless, we show that the 2.3 nm-sized CdSe NPs facilitate an eightfold increase compared with larger CdSe NPs. The obtained results were investigated using electrochemical techniques, transmission electron microscopy, and further confirmed by time-resolved spectroscopic measurements, which allow us to determine the electron tranfer rate constant (kET ) of the different configurations.

Biotechnological approaches to develop nitrogen-fixing cereals: A review

Agricultural yields are often limited by nitrogen (N) availability, especially in countries of the developing world, whereas in industrialized nations the application of chemical N fertilizers has reached unsustainable levels that have resulted in severe environmental consequences. Finding alternatives to inorganic fertilizers is critical for sustainable and secure food production. Although gaseous nitrogen (N2) is abundant in the atmosphere, it cannot be assimilated by most living organisms. Only a selected group of microorganisms termed diazotrophs, have evolved the ability to reduce N2 to generate NH3 in a process known as biological nitrogen fixation (BNF) catalysed by nitrogenase, an oxygen-sensitive enzyme complex. This ability presents an opportunity to improve the nutrition of crop plants, through the introduction into cereal crops of either the N fixing bacteria or the nitrogenase enzyme responsible for N fixation. This review explores three potential approaches to obtain N-fixing cereals: (a) engineering the nitrogenase enzyme to function in plant cells; (b) engineering the legume symbiosis into cereals; and (c) engineering cereals with the capability to associate with N-fixing bacteria.

(Keynote) Engineering Cyanobacteria for Nitrogen Reduction

Ammonia is currently the second most widely produced chemical in the world (second to sulfuric acid). Most ammonia (over 66%) is now produced through the Haber−Bosch process, consuming approximately 5% of the global natural gas production and contributes to over 3% of global carbon dioxide emissions. In an effort to develop an electrochemical alternative, we have engineered cyanobacterium to perform nitrogen fixation under ambient conditions without consuming natural gas, in stark contrast to the Haber-Bosch process that requires extreme temperatures and pressures to the same end. To realize the heterologous expression of nitrogenase, we transformed the cyanobacterium S. elongatus PCC 7942 with the minimal nitrogen fixation (nif) gene cluster. Nitrogenase can reduce N2 to NH3 at mild conditions (< 40 oC, atmospheric pressure). There are three known types of nitrogenase, in which the central composition of their active-site metalloclusters are molybdenum(Mo), vanadium (V), and iron (Fe), respectively. Most biological nitrogen fixation is catalyzed by the Mo-based nitrogenase enzyme. Biosynthesis of Mo-nitrogenase generally requires a large number of nif genes. However, the transformation and transcription of such a larger set of gene cluster into the cyanobacterium is challenging. In this work, a minimal nif cluster with reduced genetic complexity was chosen to accomplish the transformation of the cyanobacterium S. elongatus PCC 7942. The cyanobacterium S. elongatus PCC 7942 was transformed with nif genes and it was shown to be able to express active nitrogenase. The engineered S. elongatus PCC 7942 was immobilized on an electrode to accelerate the microbial conversion of nitrogen into ammonium using electrochemical driving forces. Methyl viologen (MV) was added as an electron mediator to shuttle electrons from the electrode to the enzyme active center, since MV can go through the cell membrane and deliver continuous electrons to nitrogenase.

Unravelling unknown cyanobacteria diversity linked with HCN production

Cyanobacteria are ecologically versatile microorganisms, occupying diverse habitats, from terrestrial caves to coastal shores and from brackish lakes to thermal springs. Cyanobacteria have also been linked with hydrogen cyanide (HCN), mainly for their ability to catabolize HCN by the nitrogenase enzyme. In this context, we sampled disparate environments, spanning from Canary Islands and Iceland to Estonia and Cyprus. Eighty-one (81) strains were isolated and characterised with taxonomic indices and molecular markers (16S-23S rRNA region and cpcBA region), whilst their ability to produce HCN was evaluated. This approach resulted in the description of five new genera (Speleotes, Haliplanktos, Olisthonema, Speos, and Iphianassa) and their type species (S. anchialus, H. antonyquinny, O. eestii, S. fyssassi, I. zackieohae) representing Chroococcales, Chroococcidiopsales, Oscillatoriales, Synechococcales, and Nostocales orders, respectively. We also found unique lineages inside the genera Komarekiella, Stenomitos, Cyanocohniella, and Nodularia, describing four new species (K. chia, S. pantisii, C. hyphalmyra, N. mediterannea). We report for the first time a widespread production of HCN amongst different taxa and habitats. Epilithic lifestyle, where cyanobacteria are more vulnerable to grazers, had the largest relative frequency in HCN production. In this work, we show novel cyanobacteria diversity from various habitats, including an unexplored anchialine cave, and possible correlation of cyanobacteria chemo- with species diversity, which may have implications on strategic focusing of screening programs on underexploited taxa and/or habitats.