Nitrogen-fixing Cyanobacteria Research Articles

Spatial heterogeneity in the photobiology of phototrophs—questions and methods

Photosynthesis which harvests energy from the Sun, whether it occurs in prokaryotes or eukaryotes, is a key biological process that ultimately nourishes the biosphere. The molecular efficiencies of the photo-physical and physiological processes are intricately tied not only to the photo-physics/enzymatic kinetics of the proteins involved, but also to their spatial co-localization in membrane microdomains or in cell compartments (e.g., in membrane-less organelles). Similar heterogeneity in function can be found also between cells in isogenic cell cultures (phenotypic heterogeneity) or in filaments of phototrophic cells (e.g., heterocysts/vegetative cells in nitrogen fixing cyanobacteria). This review paper delves into the connection between the spatial (co)-localization of biomolecules (lipids, RNA, DNA, proteins, membranes compartments) and their functionality in situ. We highlight recent methodological advances in the field (e.g., super-resolution microscopy, Raman micro-spectroscopy, nanoSIMS, microsensors) and showcase applications of these methods in understanding heterogeneity on single-cell and on population-scale level. This paper thus aims to highlight the avenues that will help to unravel the molecular, cellular and ecological mechanisms in photobiology by combining up-to-date microscopy techniques with more traditional functional approaches.

Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

The cumulative impact of temperature and nitrogen availability on the potential nitrogen fixation and extracellular polymeric substances secretion by Dolichospermum

Nitrogen-fixing cyanobacteria not only cause severe blooms but also play an important role in the nitrogen input processes of lakes. The production of extracellular polymeric substances (EPS) and the ability to fix nitrogen from the atmosphere provide nitrogen-fixing cyanobacteria with a competitive advantage over other organisms. Temperature and nitrogen availability are key environmental factors in regulating the growth of cyanobacteria. In this study, Dolichospermum (formerly known as Anabaena) was cultivated at three different temperatures (10°C, 20°C, and 30°C) to examine the impact of temperature and nitrogen availability on nitrogen fixation capacity and the release of EPS. Initially, confocal laser scanning microscopy (CLSM) and the quantification of heterocysts at different temperatures revealed that lower temperatures (10°C) hindered the differentiation of heterocysts under nitrogen-deprived conditions. Additionally, while heterocysts inhibited the photosynthetic activity of Dolichospermum, the secretion of EPS was notably affected by nitrogen limitation, particularly at 30°C. Finally, real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression of nitrogen-utilizing genes (ntcA and nifH) and EPS synthesis-related genes (wzb and wzc). The results indicated that under nitrogen-deprived conditions, the expression of each gene was upregulated, and there was a significant correlation between the upregulation of nitrogen-utilizing and EPS synthesis genes (P < 0.05). Our findings suggested that Dolichospermum responded to temperature variation by affecting the formation of heterocysts, impacting its potential nitrogen fixation capacity. Furthermore, the quantity of EPS released was more influenced by nitrogen availability than temperature. This research enhances our comprehension of interconnections between nitrogen deprivation and EPS production under the different temperatures.

Nitrogen-fixing cyanobacteria enhance microbial carbon utilization by modulating the microbial community composition in paddy soils of the Mollisols region

Nitrogen-fixing cyanobacteria (NFC) are photosynthetic prokaryotic microorganisms capable of nitrogen fixation. They can be used as biofertilizers in paddy fields, thereby improving the rice tillering capacity and yield. To reveal the microbiological mechanisms by which nitrogen-fixing cyanobacteria alter soil carbon storage, we conducted a field experiment using NFC as a partial substitute for nitrogen fertilizer in paddy fields in the Sanjiang Plain of Northeast China's Mollisols region. Using metagenomic sequencing technology and Biolog Ecoplate™ carbon matrix metabolism measurements, we explored the changes in the soil microbial community structure and carbon utilization in paddy fields. The results indicated that the replacement of nitrogen fertilizer with NFC predisposed the soil microbial community to host a great number of copiotrophic bacterial taxa, and Proteobacteria and Actinobacteria were closely associated with the metabolism of soil carbon sources. Moreover, through co-occurrence network analysis, we found that copiotrophic bacteria clustered in modules that were positively correlated with the metabolic level of carbon sources. The addition of NFC promoted the growth of copiotrophic bacteria, which increased the carbon utilization level of soil microorganisms, improved the diversity of the microbial communities, and had a potential impact on the soil carbon stock. The findings of this study are helpful for assessing the impact of NFC on the ecological function of soil microbial communities in paddy fields in the black soil area of Northeast China, which is highly important for promoting sustainable agricultural development and providing scientific reference for promoting the use of algal-derived nitrogen fertilizers.

The commercial potential of Aphanizomenon flos-aquae, a nitrogen-fixing edible cyanobacterium

AbstractAphanizomenon flos-aquae (AFA) is a Gram-negative nitrogen-fixing freshwater filamentous cyanobacterium with a rich nutritional profile that is approved for human consumption by both the Food and Drug Administration and the European Food and Safety Authorities. It has a high protein content (60-70%) and contains numerous vitamins, minerals and trace elements together with several high-value compounds with nutraceutical properties, such as C-phycocyanin and β-phenylethylamine. 500-1000 t of AFA dry biomass are currently wild harvested from natural blooms that occur seasonally in Klamath Lake, Oregon, USA, and distributed as a nutritional supplement worldwide. The requirements and unreliability of wild harvesting, owing to the dependence of AFA growth on environmental conditions and potential contamination by microcystin toxins, threaten the availability of biomass supply and restricts commercial expansion. In this review we propose AFA cultivation in open ponds or closed photobioreactors to obtain a reliable production of unialgal biomass to resolve the supply issue and enhance AFA as a feedstock for specific high-value by-products. Furthermore, we explore potential strategies for maximising overall yield and seasonal robustness by using a synergistic combination of cultivation and wild harvesting. Controlled AFA cultivation would also facilitate the use of genetic manipulation to generate bespoke strains with improved commercial applications, such as increasing the cyanobacterium’s nitrogen-fixation rate to enhance its value as a biofertiliser. Ultimately, realising the untapped biotechnological potential of AFA requires a better understanding of its fundamental biology, robust methodologies for laboratory and large-scale cultivation, and the development of AFA-specific genetic engineering techniques.

Nanoplastics impair growth and nitrogen fixation of marine nitrogen-fixing cyanobacteria

Nanoplastics pollution is a growing environmental problem worldwide. Recent research has demonstrated the toxic effects of nanoplastics on various marine organisms. However, the influences of nanoplastics on marine nitrogen-fixing cyanobacteria, a critical nitrogen source in the ocean, remained unknown. Here, we report that nanoplastics exposure significantly reduced growth, photosynthetic, and nitrogen fixation rates of Crocosphaera watsonii (a major marine nitrogen-fixing cyanobacterium). Transcriptomic analysis revealed that nanoplastics might harm C. watsonii via downregulation of photosynthetic pathways and DNA damage repair genes, while genes for respiration, cell damage, nitrogen limitation, and iron (and phosphorus) scavenging were upregulated. The number and size of starch grains and electron-dense vacuoles increased significantly after nanoplastics exposure, suggesting that C. watsonii allocated more resources to storage instead of growth under stress. We propose that nanoplastics can damage the cell (e.g., DNA, cell membrane, membrane-bound transporters, etc.), inhibit N2 and CO2 fixation, and hence lead to nutrient limitation and impaired growth. Our findings suggest the possibility that nanoplastics pollution could reduce the new nitrogen input and hence affect the productivity in the ocean. The impact of nanoplastics on marine nitrogen fixation and productivity should be considered when predicting the ecosystem response and biogeochemical cycling in the changing ocean.

Crocosphaera watsonii - A widespread nitrogen-fixing unicellular marine cyanobacterium.

Crocosphaera watsonii is a unicellular N2-fixing (diazotrophic) cyanobacterium observed in tropical and subtropical oligotrophic oceans. As a diazotroph, it can be a source of bioavailable nitrogen (N) to the microbial community in N-limited environments, and this may fuel primary production in the regions where it occurs. Crocosphaera watsonii has been the subject of intense study, both in culture and in field populations. Here, we summarize the current understanding of the phylogenetic and physiological diversity of C. watsonii, its distribution, and its ecological niche. Analysis of the relationships among the individual Crocosphaera species and related free-living and symbiotic lineages of diazotrophs based on the nifH gene have shown that the C. watsonii group holds a basal position and that its sequence is more similar to Rippkaea and Zehria than to other Crocosphaera species. This finding warrants further scrutiny to determine if the placement is related to a horizontal gene transfer event. Here, the nifH UCYN-B gene copy number from a recent synthesis effort was used as a proxy for relative C. watsonii abundance to examine patterns of C. watsonii distribution as a function of environmental factors, like iron and phosphorus concentration, and complimented with a synthesis of C. watsonii physiology. Furthermore, we have summarized the current knowledge of C. watsonii with regards to N2 fixation, photosynthesis, and quantitative modeling of physiology. Because N availability can limit primary production, C. watsonii is widely recognized for its importance to carbon and N cycling in ocean ecosystems, and we conclude this review by highlighting important topics for further research on this important species.

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120.

Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of Anabaena, the analysis of the extracellular metabolites and proteins of a furC-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of Anabaena. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of furC-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and amiC gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including amiC1 and amiC2, could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the furC-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in furC-overexpressing cells, namely 1,6-anhydro-N-acetyl-β-D-muramic acid (anhydroMurNAc) and its associated disaccharide (β-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.IMPORTANCECyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions. Anabaena sp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator in Anabaena, which modulates the response to several stresses. Here, we show that furC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD, and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Effect of salinity stress on growth, chlorophyll, antioxidant enzymes and nutrient content in Azolla spp.

Azolla is an aquatic fern that has a symbiotic association with nitrogen-fixing cyanobacteria. It is mainly used as a biofertilizer in rice; however, its potential under salt-affected rice cultivated area was compromised. Therefore, the present study was undertaken to understand the effect of salinity stress on morpho-physiological, biochemical characteristics, photosynthetic efficacy, nutrient and High Affinity Potassium Transporter (HKT) genes in Azolla. The results indicated that out of 102, 8 Azolla (A. microphylla, BLCC 5, BLCC 18, BLCC 28, Pa Car WTY, R 18, R 54 and R 59) were found tolerant to 80 mM NaCl. The best species for salt tolerant (80 mM NaCl) was A. microphylla, whereas the least-tolerant was A. rubra. Fresh biomass production, frond length and width in A. microphylla were significantly (p < 0.05) higher in A. microphylla than A. rubra in both 40 and 80 mM NaCl. Moreover, chlorophyll a/b ratio, carotenoids and chlorophyll fluorescence (CHF)-derived FO, Fm, Fv/Fm and root architecture (root length, average root diameter, root volume, projectile and surface area) were higher in A. microphylla than A. rubra under 40 and 80 mM NaCl. Contents of Na+ and Ca2+ increased in both A. microphylla and A. rubra, which can interfere with the uptake of essential macronutrients; however, these were accumulated comparatively less in A. microphylla than A. rubra, whereas a reverse trend was observed in cellular accumulation of K+ content. A. microphylla had higher superoxide dismutase (SOD), ascorbate peroxidase (APX), and proline activities in 40 and 80 mM NaCl than A. rubra. For the first time, twenty six HKT primers were designed as a molecular marker to identify salt-tolerant Azolla. Out of these, three HKT primers (Req 6, Aeq14, and Aeq16) were amplified in A. microphylla under NaCl stress, while their amplifications were not observed in A. rubra (salt susceptible). In A. microphylla, the expression of the Req 6 (HKT) gene were more under NaCl stress. Moreover, further research is needed to discover and validate the biochemical and molecular processes that confer salinity tolerance in Azolla plants.

Physiological Characteristics and Nitrogen and Iron Utilization of Marine Polycultures and Bundles of Hair Algae

In response to the problem of iron limitation in nitrogen-fixing cyanobacteria, this study explores the potential for symbiotic and mutually beneficial community survival of Polychaeta and Trichoderma. The presence of the two elements and their effects on the survival of the cyanobacteria were first analysed in the context of nitrogen and iron use and cycling in the ocean. The physiological characteristics of the two algae were also analysed in relation to environmental factors such as light, ph and temperature in the marine water column. 730The results of the incubation experiments showed that the filtrate of Trichoderma reesei had a significant effect on the growth of Polycoccus, with the OD730 rising to 0.0372 within 72 hours of the addition of Trichoderma reesei. The addition of the wild-type iron carrier secretion of G. globulus also promoted the growth rate and nitrogen fixation efficiency of the algae. The symbiotic relationship between the nitrogen and iron utilisation of the algae was shown to contribute to the growth and nitrogen fixation activities of both species.

Aluminum-induced changes in the net carbon fixation and carbon decomposition of a nitrogen-fixing cyanobacterium Trichodesmium erythraeum

Aluminum-induced changes in the net carbon fixation and carbon decomposition of a nitrogen-fixing cyanobacterium Trichodesmium erythraeum

Symbiosis between cyanobacteria and plants: from molecular studies to agronomic applications.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

Влияние навозных стоков на почвенные фототрофные микроорганизмы

Soil phototrophic microorganisms (algae, cyanobacteria) play a very important role in functioning of anthropogenic systems. They take an active part in soil self-purification by directly absorbing biogenic elements, as well as by stimulating the activity of mineralizing microorganisms at the expense of disengaged oxygen. The research was carried out at a plough field with sod-podzol soil situated near a large pig-breeding farm. Neutralization took place due to natural microbiological processes during the time of disgorging the liquid fraction in the lagoons. Long-term application of manure runoff from pig-breeding farms into plough soil had an impact on species diversity of phototrophic microorganisms, which was considered in the research. Groups of species tolerant to the impact formed in the soil in course of long-term application of liquid manure fraction. The influence of long-term runoff of manure from pig breeding farms on specious diversity of soil phototrophic microorganisms was assessed. In plough soils affected by liquid manure runoff a group of species tolerant to this impact has formed. Their basis is represented by green algae: Klebsormidium flaccidum, K. nitens, Chlorococcum infusionum, Chlorella vulgaris, as well as by the heterocyst-free cyanobacteria Phormidium autumnale and the diatom alga Hantzschia amphioxys. On the surface of plough soil these species make microscopically marked films, i. e. soil “blooming” takes place. General species diversity of microphototrophs in soil of the experimental site is lower than that of the control site. Jaccard coefficient of the experimental and control sites of the field is 47.8%. Nitrogen-fixing cyanobacteria are not represented in the microphototrophic community; it indicates that the natural microcoenosis is damaged in the plough soils and nitrogen content is high.

Cyanobacteria: Isolation, Purification and Principles

The primary means of nutrient uptake for cyanobacteria is oxygen-producing photosynthesis. Their ecological variety is astounding; they occupy a very wide range of lighted ecological niches in terrestrial, marine, and freshwater habitats. Despite this apparent metabolic consistency, they exhibit tremendous phylogenetic diversity. The fact that cyanobacteria have certain physiological and metabolic traits that are exclusively seen in prokaryotes significantly broadens this spectrum. The capacity to fix nitrogen in an aerobic manner under light is a special characteristic. Apart from Gonotheca species, all aerobic, nitrogen-fixing cyanobacteria—a structurally diverse group—produce heterocyst, the highly specialised cells that allow them to fix nitrogen efficiently in a fully aerobic environment by preventing the oxygen-sensitive enzyme nitrogenase from being inactivated quickly in vivo (Hazelton, 1978; Stanier and Cohen-Bazire, 1978; Stewart, Haystead, and Pearson, 1969). The same quantities of cyanobacteria could be isolated from freshwater using a technique using nutrient-saturated glass fibre filters, but the quantity of accompanying heterotrophic bacteria was reduced by 2- to 15-fold. a broad-spectrum antibiotic called imipenem. In comparison to some other Plactam antibiotics, the B-lactam antibiotic that inhibits peptidoglycan biosynthesis, was more effective at lowering the levels of heterotrophic bacterial contaminants associated with newly isolated cyanobacteria to a point that made it easier to grow axenic cyanobacterial cultures.

A fluorescence method to detect cyanophycin in the symbiotic cyanobacterium, Anabaena azollae

ABSTRACT The cyanophycin content of the heterocystous nitrogen-fixing symbiotic cyanobacterium, Anabaena azollae, which inhabits an ovoid cavity in the dorsal leaf lobes of the fern, Azolla filiculoides, is seldom analyzed. To study the cyanophycin content in vegetative cells and heterocysts of A. azollae, we used three fluorochromes: aluminum trichloride, lead citrate and Wilson citroboric solution and Coomassie brilliant blue. Blue and yellow fluorescence were emitted from the polar nodes and cytoplasm cyanophycin granules of the heterocysts when stained with the three fluorochromes. The cyanophycin observed without staining or with Coomassie brilliant blue staining did not alter the results obtained using the fluorochromes. We found that aluminum trichloride, lead acetate and Wilson citroboric solution could be used to detect cyanophycin.

An integrated approach for the phycoremediation of Pb(II) and the production of biofertilizer using nitrogen-fixing cyanobacteria.

In recent years, growing attention has been directed toward the phycoremediation of heavy metals from bodies of water; however, many challenges remain. The nitrogen requirements for algal growth in nutrient-poor waters can lead to substantial costs. Moreover, proper management of the metal-loaded biomass is a concern. This study assessed the performance of two nitrogen-fixing cyanobacteria, Anabaena sp. and Nostoc muscorum, in treating Pb(II)-contaminated water without nitrogen under batch and fed-batch modes, as well as the subsequent utilization of the produced biomass as a biofertilizer. After 12 days of the batch mode with initial Pb(II) concentrations of 10, 20, 35, and 60mg/L, Pb(II) removal efficiencies were 98.90%, 98.95%, 97.20%, and 84.98% by Anabaena sp. and 88.00%, 73.10%, 54.54%, and 26.83% by N. muscorum, respectively. Anabaena sp. sustained growth and Pb(II) removal under the fed-batch mode by adjusting hydraulic retention time based on the influent Pb(II) concentration. Decontamination of the metal-loaded Anabaena sp. biomass was performed and resulted in a Pb(II) desorption of 93%. The desorbed Anabaena sp. extract provided the nutrient requirements for Chlorella vulgaris. The proposed strategy provides simultaneous Pb(II) bioremediation and biofertilizer production in a system driven by light energy, atmospheric N2, and CO2.

Investigating the Unique Ability of Trichodesmium To Fix Carbon and Nitrogen Simultaneously Using MiMoSA.

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle, Trichodesmium is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen. Here, we use the advanced modeling framework called multiscale multiobjective systems analysis (MiMoSA) to investigate how Trichodesmium erythraeum can reduce dimolecular nitrogen to ammonium in the presence of oxygen. Our simulations indicate that nitrogenase inhibition is best modeled as Michealis-Menten competitive inhibition and that cells along the filament maintain microaerobia using high flux through Mehler reactions in order to protect nitrogenase from oxygen. We also examined the effect of location on metabolic flux and found that cells at the end of filaments operate in distinctly different metabolic modes than internal cells despite both operating in a photoautotrophic mode. These results give us important insight into how this species is able to operate photosynthesis and nitrogen fixation simultaneously, giving it a distinct advantage over other diazotrophic cyanobacteria because they can harvest light directly to fuel the energy demand of nitrogen fixation. IMPORTANCE Trichodesmium erythraeum is a marine cyanobacterium responsible for approximately half of all biologically fixed nitrogen, making it an integral part of the global nitrogen cycle. Interestingly, unlike other nitrogen-fixing cyanobacteria, Trichodesmium does not use temporal or spatial separation to protect nitrogenase from oxygen poisoning; instead, it operates photosynthesis and nitrogen fixation reactions simultaneously during the day. Unfortunately, the exact mechanism the cells utilize to operate carbon and nitrogen fixation simultaneously is unknown. Here, we use an advanced metabolic modeling framework to investigate and identify the most likely mechanisms Trichodesmium uses to protect nitrogenase from oxygen. The model predicts that cells operate in a microaerobic mode, using both respiratory and Mehler reactions to dramatically reduce intracellular oxygen concentrations.

Primer design for the amplification of the ammonium transporter genes from the uncultured haptophyte algal species symbiotic with the marine nitrogen-fixing cyanobacterium UCYN-A1.

The multiple symbiotic partnerships between closely related species of the haptophyte algae Braarudosphaera bigelowii and the nitrogen-fixing cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) contribute importantly to the nitrogen and carbon cycles in vast areas of the ocean. The diversity of the eukaryotic 18S rDNA phylogenetic gene marker has helped to identify some of these symbiotic haptophyte species, yet we still lack a genetic marker to assess its diversity at a finer scale. One of such genes is the ammonium transporter (amt) gene, which encodes the protein that might be involved in the uptake of ammonium from UCYN-A in these symbiotic haptophytes. Here, we designed three specific PCR primer sets targeting the amt gene of the haptophyte species (A1-Host) symbiotic with the open ocean UCYN-A1 sublineage, and tested them in samples collected from open ocean and near-shore environments. Regardless of the primer pair used at Station ALOHA, which is where UCYN-A1 is the pre-dominant UCYN-A sublineage, the most abundant amt amplicon sequence variant (ASV) was taxonomically classified as A1-Host. In addition, two out of the three PCR primer sets revealed the existence of closely-related divergent haptophyte amt ASVs (>95% nucleotide identity). These divergent amt ASVs had higher relative abundances than the haptophyte typically associated with UCYN-A1 in the Bering Sea, or co-occurred with the previously identified A1-Host in the Coral Sea, suggesting the presence of new diversity of closely-related A1-Hosts in polar and temperate waters. Therefore, our study reveals an overlooked diversity of haptophytes species with distinct biogeographic distributions partnering with UCYN-A, and provides new primers that will help to gain new knowledge of the UCYN-A/haptophyte symbiosis.

Emergence of hydrocarbon-degrading bacteria in crude oil-contaminated soil in a hyperarid ecosystem: Effect of phosphate addition and augmentation with nitrogen-fixing cyanobacteria on oil bioremediation

Hyperarid hot deserts impose limiting conditions for microbial activity, thus delaying crude oil biodegradation resulting from accidental spills. This study examined the emergence of oil-degrading bacterial species in the soil of a hyperarid area in the southern Negev desert, Israel, which was exposed to a major oil spill. Amplicon sequencing of the 16S rRNA gene showed dominance of the phyla Proteobacteria and Actinobacteria in both contaminated and control soils. Significant differences were seen in the abundance of the phylum Firmicutes. Classes Actinobacteria, Gammaproteobacteria and Bacilli dominated the oil-contaminated soil. Bacterial species capable of degrading aromatic and aliphatic oil constituents were isolated from the contaminated soil. Contaminated soil microcosms were amended separately with nitrogen, phosphorus, or nitrogen-fixing cyanobacteria (NFC) biomass; the highest reduction in total petroleum hydrocarbons was observed in the phosphorus-supplemented soil. The abundance of alkB gene, encoding alkane monooxygenase, increased significantly in the control, NFC and phosphate treatments, but not in the nitrogen treatment. However, nahA abundance (coding for naphthalene dioxygenase) was only considerably increased with the phosphorus amendment. While nitrogen amendment via NFC bioaugmentation is a sustainable and novel biostimulation method, phosphorus amendment significantly accelerates the biodegradation of crude oil under extremely arid conditions.