Albertania and Egbenema gen. nov. from Nigeria and the United States, expanding biodiversity in the Oculatellaceae (cyanobacteria).

Knowledge of the tropical terrestrial cyanobacterial flora from the African continent is still limited. Of 31 strains isolated from soil and subaerial samples collected in Lagos State, Nigeria, three were found to be in the Oculatellaceae, including two species in a new genus. Subsequently, isolates from microbial mats in White Sands National Park in New Mexico, United States, and from a rock near the ocean in Puerto Rico, United States, were found to belong to the new genus as well. Cyanobacterial isolates were characterized microscopically, sequenced for the 16S rRNA gene and associated ITS region, and phylogenetically analyzed. Egbenema gen. nov., with three new species, as well as two new species of Albertania were differentiated from all other Oculatellaceae. Both genera belong to a supported clade within the Oculatellaceae that includes Trichotorquatus and Komarkovaea. The two new species of Albertania, A. egbensis and A. latericola, were from the same sample, but were evolutionarily separate based on 16S rRNA gene phylogenies, percent identity below the 98.7% threshold, and ITS rRNA percent dissimilarity >7.0%. Egbenema aeruginosum gen. et sp. nov. was phylogenetically separated from Trichotorquatus and Albertania but was in a clade with other strains belonging to Egbenema. The two Egbenema strains from the United States are here named Egbenema epilithicum sp. nov. and Egbenema gypsiphilum sp. nov. Our results support the hypothesis that further species discoveries of novel cyanobacteria will likely be made in soils and subaerial habitats, as these habitats continue to be studied, both in tropical and temperate biomes.

The Enzyme Lysine Malonylation of Calvin Cycle and Gluconeogenesis Regulated Glycometabolism in Nostoc flagelliforme to Adapt to Drought Stress.

Lysine malonylation (Kmal) is an evolutionarily conserved post-translational modification (PTM) that has been demonstrated to be involved in cellular and organismal metabolism. However, the role that Kmal plays in response to drought stress of the terrestrial cyanobacteria N. flagelliforme is still unknown. In this study, we performed the first proteomic analysis of Kmal in N. flagelliforme under different drought stresses using LC-MS/MS. In total, 421 malonylated lysine residues were found in 236 different proteins. GO and KEGG enrichment analysis indicated that these malonylated proteins were highly enriched in several metabolic pathways, including carbon metabolism and photosynthesis. Decreased malonylation levels were found to hinder the reception and transmission of light energy and CO2 fixation, which led to a decrease in photosynthetic activity. Kmal was also shown to inhibit the flux of the TCA cycle and activate the gluconeogenesis pathway in response to drought stress. Furthermore, malonylated antioxidant enzymes and antioxidants were synergistically involved in reactive oxygen species (ROS) scavenging. Malonylation was involved in lipid degradation and amino acid biosynthesis as part of drought stress adaptation. This work represents the first comprehensive investigation of the role of malonylation in dehydrated N. flagelliforme, providing an important resource for understanding the drought tolerance mechanism of this organism.

Influence of wettability and surface design on the adhesion of terrestrial cyanobacteria to additive manufactured biocarriers.

Productive biofilms are gaining growing interest in research due to their potential of producing valuable compounds and bioactive substances such as antibiotics. This is supported by recent developments in biofilm photobioreactors that established the controlled phototrophic cultivation of algae and cyanobacteria. Cultivation of biofilms can be challenging due to the need of surfaces for biofilm adhesion. The total production of biomass, and thus production of e.g. bioactive substances, within the bioreactor volume highly depends on the available cultivation surface. To achieve an enlargement of surface area for biofilm photobioreactors, biocarriers can be implemented in the cultivation. Thereby, material properties and design of the biocarriers are important for initial biofilm formation and growth of cyanobacteria. In this study, special biocarriers were designed and additively manufactured to investigate different polymeric materials and surface designs regarding biofilm adhesion of the terrestrial cyanobacterium Nostoc flagelliforme (CCAP 1453/33). Properties of 3D-printed materials were characterized by determination of wettability, surface roughness, and density. To evaluate the influence of wettability on biofilm formation, material properties were specifically modified by gas-phase fluorination and biofilm formation was analyzed on biocarriers with basic and optimized geometry in shaking flask cultivation. We found that different polymeric materials revealed no significant differences in wettability and with identical surface design no significant effect on biomass adhesion was observed. However, materials treated with fluorination as well as optimized biocarrier design showed improved wettability and an increase in biomass adhesion per biocarrier surface.

Assessment of Common Cyanotoxins in Cyanobacteria of Biological Loess Crusts.

Cyanotoxins are a diverse group of bioactive compounds produced by cyanobacteria that have adverse effects on human and animal health. While the phenomenon of cyanotoxin production in aquatic environments is well studied, research on cyanotoxins in terrestrial environments, where cyanobacteria abundantly occur in biocrusts, is still in its infancy. Here, we investigated the potential cyanotoxin production in cyanobacteria-dominated biological loess crusts (BLCs) from three different regions (China, Iran, and Serbia) and in cyanobacterial cultures isolated from the BLCs. The presence of cyanotoxins microcystins, cylindrospermopsin, saxitoxins, and ß-N-methylamino-L-alanine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, while the presence of cyanotoxin-encoding genes (mcyE, cyrJ, sxtA, sxtG, sxtS, and anaC) was investigated by polymerase chain reaction (PCR) method. We could not detect any of the targeted cyanotoxins in the biocrusts or the cyanobacterial cultures, nor could we amplify any cyanotoxin-encoding genes in the cyanobacterial strains. The results are discussed in terms of the biological role of cyanotoxins, the application of cyanobacteria in land restoration programs, and the use of cyanotoxins as biosignatures of cyanobacterial populations in loess research. The article highlights the need to extend the field of research on cyanobacteria and cyanotoxin production to terrestrial environments.

The Overlooked Genetic Diversity in the Dryland Soil Surface-Dwelling Cyanobacterium Nostoc flagelliforme as Revealed by the Marker Gene wspA.

Biodiversity is recognized to be relatively low in the dryland ecosystem. However, we might overlook the accumulating genetic variation in those dryland micro-populations, which should eventually increase the dryland biodiversity. In the xeric steppes of western and northwestern China, there are two soil surface-dwelling and genetically close cyanobacterial species, Nostoc commune and Nostoc flagelliforme. They respectively exhibit lamellate and filamentous colony shapes. Their individual colony is consisted of hundreds of trichomes and the common exopolysaccharide matrix. N. flagelliforme is exclusively distributed in the dryland and supposed to be evolved from N. commune. We previously reported that the morphological diversity of N. flagelliforme colonies was very limited, being either cylindrical or strip-like. In this communication, we performed single-nucleotide polymorphism (SNP) analysis of the marker gene wspA as well as phylogenetic analysis of the WspA protein in N. flagelliforme colonies to gain insights into its genetic diversity. SNP analysis suggested that there existed plentiful nucleotide variations in the individual colonies and meanwhile these variations shared certain evolutionary regularity. Phylogenetic analysis of the deduced proteins from the cloned wspA sequences suggested that the relatively regular variations were possibly dispersed in the N. flagelliforme populations of different regions. Thus, these results presented a scenario of the underestimated genetic diversity hidden behind the limited morphotype of dryland cyanobacteria. Maybe, we can consider the individual cyanobacterial colony as a potential biodiversity pool in the drylands.

Development of a lightweight multi-skin sheet photobioreactor for future cultivation of phototrophic biofilms on facades.

This article covers the development of a novel emerse photobioreactor (ePBR), using a polycarbonate multi-skin sheet (MSS), to cultivate terrestrial cyanobacteria as surface-associated phototrophic biofilms in an aerosol-based cultivation process. The aerosol, generated by ultrasonic transduction, moistens and nourishes the biofilm inside the multi-skin sheet emerse photobioreactor (MSSePBR). Advantages of the MSSePBR, such as its low weight design and reduced water consumption due to the usage of aerosol, simplify the development for future facade bioreactors. To develop the MSSePBR, surface roughness, static contact angle and luminous transmittance were investigated to characterize the properties of the cultivation surface for phototrophic cultivation. The polymeric MSS showed good luminous transmittance and proofed its optical suitability for the cultivation of terrestrial cyanobacteria. Using the MSSePBR, the terrestrial cyanobacteria Coleofasciculus chthonoplastes and Trichocoleus sociatus were cultivated with either ambient air, air with increased CO2 content or flue gas. The cultivation of terrestrial cyanobacteria showed higher productivities for biomass in the MSSePBR than in suspended systems. Cultivation with increased CO2 contents and flue gas was possible, thus a combination with flue gas treatment is feasible. An up-scaled prototype of the MSSePBR was introduced to show the possibilities for future industrial-sized and facade applications.

Geographical Variability of Mineral Elements and Stability of Restrictive Mineral Elements in Terrestrial Cyanobacteria Across Gradients of Climate, Soil, and Atmospheric Wet Deposition Mineral Concentration.

Terrestrial cyanobacteria Nostoc commune is an ideal species to study the geographical variation of mineral elements of soil cyanobacteria at the species level. Here, we first address the following questions: (1) from where are these mineral elements, (2) are there geographical variations for these mineral elements, and if so, (3) which environmental factors drive the geographical variation of these mineral elements? Second, we tested whether the soil cyanobacterial mineral elements followed the "restrictive element stability hypothesis" of higher plants. Finally, we explored the effect of mineral geographic variation on ecological adaptation of soil cyanobacteria. We collected N. commune samples across gradients of climate, soil, and atmospheric wet deposition mineral concentration in mainland China. We measured fifteen minerals, including five macroelements (N, Ca, K, Fe, P), five microelements (Mn, Zn, Cu, Co, Se), and five heavy metals (Pb, Cr, As, Cd, Hg). We found that five elements (P, Cu, Zn, Co, Pb) had significant geographical variation. They increased as the distance from the equator increased and decreased as the distance from the prime meridian increased. Mean annual precipitation and mean annual temperature explained most of the variation. We did not find any significant correlations between the mineral element contents in N. commune and the minerals in soil and rainfall, except for P. There was no significant correlation between the variation coefficients of different elements and their actual detected contents and their potential physiological required contents. The statistical results of our experiment did not support the "restrictive element stability hypothesis." We speculated that net accumulation of mineral elements in cyanobacterial cells and extracellular polysaccharides (EPS) might play an important role for terrestrial cyanobacteria in the adaptation to dry and cold conditions.

The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant.

Environmental abiotic stresses are limiting factors for less tolerant organisms, including soil plants. Abiotic stress tolerance-associated genes from prokaryotic organisms are supposed to have a bright prospect for transgenic application. The drought-adapted cyanobacterium Nostoc flagelliforme is arising as a valuable prokaryotic biotic resource for gene excavation. In this study, we evaluated the salt-tolerant function and application potential of a candidate gene drnf1 from N. flagelliforme, which contains a P-loop NTPase (nucleoside-triphosphatase) domain, through heterologous expression in two model organisms Synechocystis sp. PCC 6803 and Arabidopsis thaliana. It was found that DRNF1 could confer significant salt tolerance in both transgenic organisms. In salt-stressed transgenic Synechocystis, DRNF1 could enhance the respiration rate; slow-down the accumulation of exopolysaccharides; up-regulate the expression of salt tolerance-related genes at a higher level, such as those related to glucosylglycerol synthesis, Na⁺/H⁺ antiport, and sugar metabolism; and maintain a better K⁺/Na⁺ homeostasis, as compared to the wild-type strain. These results imply that DRNF1 could facilitate salt tolerance by affecting the respiration metabolism and indirectly regulating the expression of important salt-tolerant genes. Arabidopsis was employed to evaluate the salt tolerance-conferring potential of DRNF1 in plants. The results show that it could enhance the seed germination and shoot growth of transgenic plants under saline conditions. In general, a novel prokaryotic salt-tolerant gene from N. flagelliforme was identified and characterized in this study, enriching the candidate gene pool for genetic engineering in plants.

Application of phototrophic biofilms: from fundamentals to processes.

Biotechnological production of valuables by microorganisms is commonly achieved by cultivating the cells as suspended solids in an appropriate liquid medium. However, the main portion of these organisms features a surface-attached growth in their native habitats. The utilization of such biofilms shows significant challenges, e.g. concerning control of pH, nutrient supply, and heat/mass transfer. But the use of biofilms might also enable novel and innovative production processes addressing robustness and strength of the applied biocatalyst, for example if variable conditions might occur in the process or a feedstock (substrate) is changed in its composition. Besides the robustness of a biofilm, the high density of the immobilized biocatalyst facilitates a simple separation of the catalyst and the extracellular product, whereas intracellular target compounds occur in a concentrated form; thus, expenses for downstream processing can be drastically reduced. While phototrophic organisms feature a fabulous spectrum of metabolites ranging from biofuels to biologically active compounds, the low cell density of phototrophic suspension cultures is still limiting their application for production processes. The review is focusing on pro- and eukaryotic microalgae featuring the production of valuable compounds and highlights requirements for their cultivation as phototrophic biofilms, i.e. setup as well as operation of biofilm reactors, and modeling of phototrophic growth.

Unexpected High Diversity of Terrestrial Cyanobacteria from the Campus of the University of the Ryukyus, Okinawa, Japan.

Terrestrial cyanobacterial strains were isolated from the Nishihara campus of the University of the Ryukyus, Okinawa, Japan. The 13 sampling sites were distributed in a 200 m radius and appeared as dry, blackened stains. From these small areas, 143 cyanobacterial strains were established. The strains were divided into five morphotypes, including unicells, unicells with baeocytes, non-branching filaments, false-branching filaments, and heterocystous strains. From the strains, 105 partial 16S rRNA gene sequences were obtained and could be classified into 30 generic types. Among them, 22 unique strains and over 1100 bps of data were selected for further phylogenetic analyses. These sequences were positioned into six main clades corresponding to cyanobacterial orders: Nostocales, Chroococidiopsidales, Chroococcales, Oscillatoriales, Pleurocapsales, and Synechococcales. Almost all sequences had no identical matching data in GenBank and many of them had no closely related data. These data suggest that the terrestrial cyanobacteria are very divese even within close sampling areas, such as within the campus of the University of the Ryukyus. The established strains are not only important for classification of terrestrial cyanobacteria but also for possible application studies in the future.

A semi-continuous process based on an ePBR for the production of EPS using Trichocoleus sociatus.

Biodiversity forms the basis for a large pool of potential products and productive organisms offered by terrestrial cyanobacteria. They are stuck together by EPS (extracellular polymeric substances) that can obtain antiviral, antibacterial or anti-inflammatory substances. Most stress conditions, e.g. drought, induce the production of protective EPS or biotechnological-products for pharmaceutical application. However, the growth of a phototrophic biofilm is limited under submerged conditions. Therefore, a semi-continuous process to produce EPS by cyanobacteria was developed in an aerosol-based ePBR (emerse photobioreactor) that imitates the natural habitat of terrestrial cyanobacteria. The process consists of a growth-phase (biomass production), followed by a dry-phase (EPS-production) and a consecutive extraction. The EPS-productivities of Trichocoleus sociatus (ranging from 0.03 to 0.04gL-1d-1) were 32 times higher than described in topic-related literature. In comparison to submerge cultivations in shaking flasks, the EPS-productivities were sevenfold higher. To ensure that the extraction solvent has no influence on cell viability, a cell-vitality-test was performed. However, no statistically significant difference between the amount of living and dead cells before and after the extraction was detected. A bioactivity assay was then performed to identify antimicrobial activity within EPS extracts from emerse and submerge cultivations. The EPS revealed an antibacterial effect against gram-negative bacteria (E. coli) which was two times higher than EPS from a submerged cultivation.

Excavating abiotic stress-related gene resources of terrestrial macroscopic cyanobacteria for crop genetic engineering: dawn and challenge.

Genetically engineered (GE) crops with resistance to environmental stresses are one of the most important solutions for future food security. Numerous genes associated to plant stress resistance have been identified and characterized. However, the current reality is that only a few transgenic crops expressing prokaryotic genes are successfully applied in field conditions. These few prokaryotic genes include Agrobacterium strain CP4 EPSPS gene, Bacillus thuringiensis Cry1Ab gene and a bacterial chaperonin gene. Thus, the excavation of potentially critical genes still remains an arduous task for crop engineering. Terrestrial macroscopic cyanobacteria, Nostoc commune and Nostoc flagelliforme, which exhibit extreme resistance to desiccation stress, may serve as new prokaryotic bioresources for excavating critical genes. Recently, their marker gene wspA was heterologously expressed in Arabidopsis plant and the transgenics exhibited more flourishing root systems than wild-type plants under osmotic stress condition. In addition, some new genes associated with drought response and adaptation in N. flagelliforme are being uncovered by our ongoing RNA-seq analysis. Although the relevant work about the terrestrial macroscopic cyanobacteria is still underway, we believe that the prospect of excavating their critical genes for application in GE crops is quite optimistic.