Impact of Zero-Valent Iron Nanoparticles and Ampicillin on Adenosine Triphosphate and Lactate Metabolism in the Cyanobacterium Fremyella diplosiphon.

In cyanobacteria, the interplay of ATP and lactate dynamics underpins cellular energetics; their pronounced shifts in response to zero-valent iron (nZVI) nanoparticles and ampicillin highlight the nuanced metabolic adaptations to environmental challenges. In this study, we investigated the impact of nZVIs and ampicillin on Fremyella diplosiphon cellular energetics as determined by adenosine triphosphate (ATP) content, intracellular and extracellular lactate levels, and their impact on cell morphology as visualized by transmission electron microscopy. While a significant increase in ATP concentration was observed in 0.8 mg/L ampicillin-treated cells compared to the untreated control, a significant decline was noted in cells treated with 3.2 mg/L nZVIs. ATP levels in the combination regimen of 0.8 mg/L ampicillin and 3.2 mg/L nZVIs were significantly elevated (p < 0.05) compared to the 3.2 mg/L nZVI treatment. Intracellular and extracellular lactate levels were significantly higher in 0.8 mg/L ampicillin, 3.2 mg/L nZVIs, and the combination regimen compared to the untreated control; however, extracellular lactate levels were the highest in cells treated with 3.2 mg/L nZVIs. Visualization of morphological changes indicated increased thylakoid membrane stacks and inter-thylakoidal distances in 3.2 mg/L nZVI-treated cells. Our findings demonstrate a complex interplay of nanoparticle and antibiotic-induced responses, highlighting the differential impact of these stressors on F. diplosiphon metabolism and cellular integrity.

Lipid production and cellular changes in Fremyella diplosiphon exposed to nanoscale zerovalent iron nanoparticles and ampicillin.

With the dramatic decrease in fossil fuel stocks and their detrimental effects on the environment, renewable energy sources have gained imminent importance in the mitigation of emissions. As lipid-enriched energy stocks, cyanobacteria are the leading group of microorganisms contributing to the advent of a new energy era. In the present study, the impact of Nanofer 25 s nanoscale zero-valent iron nanoparticles (nZVIs) and ampicillin on lipid production and cellular structural changes in Fremyella diplosiphon strain B481-SD were investigated. Total lipid abundance, fatty acid methyl ester (FAME) compositions, and alkene production as detected by high-resolution two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC/TOF-MS) was significantly higher (p < 0.05) in the individual application of 0.8 mg/L ampicillin, 3.2 mg/L nZVIs, and a combined regimen of 0.8 mg/L ampicillin and 3.2 mg/L nZVIs compared to the untreated control. In addition, we identified significant increases (p < 0.05) in monounsaturated fatty acids (MUFAs) in F. diplosiphon treated with the combination regimen compared to the untreated control, 0.8 mg/L of ampicillin, and 3.2 mg/L of nZVIs. Furthermore, individual treatment with 0.8 mg/L ampicillin and the combination regimen (0.8 mg/L ampicillin + 3.2 mg/L nZVIs) significantly increased (p < 0.05) Nile red fluorescence compared to the untreated control, indicating neutral membrane lipids to be the main target of ampicillin added treatments. Transmission electron microscopy studies revealed the presence of single-layered thylakoid membranes in the untreated control, while complex stacked membranes of 5-8 layers were visualized in ampicillin and nZVI-treated F. diplosiphon. Our results indicate that nZVIs in combination with ampicillin significantly enhanced total lipids, essential FAMEs, and alkenes in F. diplosiphon. These findings offer a promising approach to augment the potential of using the strain as a large-scale biofuel agent.

Impact of Ascorbic Acid on Zero-Valent Iron Nanoparticle and UV-B Mediated Stress in the Cyanobacterium, Fremyella diplosiphon.

Fremyella diplosiphon is an ideal third-generation biofuel source due to its ability to produce transesterified lipids. While nanofer 25s zero-valent iron nanoparticles (nZVIs) improve lipid production, an imbalance between reactive oxygen species (ROS) and cellular defense can be catastrophic to the organism. In the present study, the effect of ascorbic acid on nZVI and UV-induced stress in F. diplosiphon strain B481-SD was investigated, and lipid profiles in the combination regimen of nZVIs and ascorbic acid compared. Comparison of F. diplosiphon growth in BG11 media amended with 2, 4, 6, 8, and 10 mM ascorbic acid indicated 6 mM to be optimal for the growth of B481-SD. Further, growth in 6 mM ascorbic acid combined with 3.2 mg/L nZVIs was significantly higher when compared to the combination regimen of 12.8 and 51.2 mg/L of nZVIs and 6 mM ascorbic acid. The reversal effect of UV-B radiation for 30 min and 1 h indicated that ascorbic acid restored B481-SD growth. Transesterified lipids characterized by gas chromatography-mass spectrometry indicated C16 hexadecanoate to be the most abundant fatty acid methyl ester in the combination regimen of 6 mM ascorbic acid and 12.8 mg/L nZVI-treated F. diplosiphon. These findings were supported by microscopic observations in which cellular degradation was observed in B481-SD cells treated with 6 mM ascorbic acid and 12.8 mg/L nZVIs. Our results indicate that ascorbic acid counteracts the damaging effect of oxidative stress produced by nZVIs.

Biocrude Production Using a Novel Cyanobacterium: Pilot-Scale Cultivation and Lipid Extraction via Hydrothermal Liquefaction.

The use of renewable energy to reduce fossil fuel consumption is a key strategy to mitigate pollution and climate change, resulting in the growing demand for new sources. Fast-growing proprietary cyanobacterial strains of Fremyella diplosiphon with an average life cycle of 7-10 days, and a proven capacity to generate lipids for biofuel production are currently being studied. In this study, we investigated the growth and photosynthetic pigmentation of a cyanobacterial strain (SF33) in both greenhouse and outdoor bioreactors, and produced biocrude via hydrothermal liquefaction. The cultivation of F. diplosiphon did not significantly differ under suboptimal conditions (p < 0.05), including in outdoor bioreactors with growth differences of less than 0.04 (p = 0.035) among various batches. An analysis of the biocrude's components revealed the presence of fatty acid biodiesel precursors such as palmitic acid and behenic acid, and alkanes such as hexadecane and heptadecane, used as biofuel additives. In addition, the quantification of value-added photosynthetic pigments revealed chlorophyll a and phycocyanin concentrations of 0.0011 ± 5.83 × 10-5 µg/µL and 7.051 ± 0.067 µg/µg chlorophyll a. Our results suggest the potential of F. diplosiphon as a robust species that can grow at varying temperatures ranging from 13 °C to 32 °C, while producing compounds for applications ranging from biofuel to nutritional supplements. The outcomes of this study pave the way for production-level scale-up and processing of F. diplosiphon-derived biofuels and marketable bioproducts. Fuel produced using this technology will be eco-friendly and cost-effective, and will make full use of the geographical location of regions with access to brackish waters.

Augmentation of the Photoreactivation Gene in Fremyella diplosiphon Confers UV-B Tolerance.

In spite of the enormous potential of cyanobacteria as a renewable energy source, elevated UV exposure is a major impediment to their commercial viability and productivity. Fremyella diplosiphon is a widely explored cyanobacterium with great biofuel capacity due to its high lipid content. To enhance UV stress tolerance in this species, we overexpressed the photoreactivation gene (phr A) that encodes for photolyase DNA repair enzyme in the wild type F. diplosiphon (B481-WT) by genetic transformation. Our efforts resulted in a transformant (B481-ViAnSa) with a 3808-fold increase in the phr A mRNA transcript level and enhanced growth under UV-B stress. Additionally, DNA strand breaks in the transformant were significantly lower after 12 and 16 h of UV radiation, with significantly higher dsDNA recovery in B481-ViAnSa (98.1%) compared to that in B481-WT (81.5%) at 48 h post irradiation. Photosystem II recovery time in the transformant was significantly reduced (48 h) compared to that in the wild type (72 h). Evaluation of high-value fatty acid methyl esters (FAMEs) revealed methyl palmitate, the methyl ester of hexadecenoic acid (C16:0), to be the most dominant component, accounting for 53.43% of the identified FAMEs in the transformant. Results of the study offer a promising approach to enhance UV tolerance in cyanobacteria, thus paving the way to large-scale open or closed pond cultivation for commercial biofuel production.

Antibiotic-Induced Changes in Pigment Accumulation, Photosystem II, and Membrane Permeability in a Model Cyanobacterium.

Fremyella diplosiphon is a well-studied a model cyanobacterium for photosynthesis due to its efficient light absorption potential and pigment accumulation. In the present study, the impact of ampicillin, tetracycline, kanamycin, and cefotaxime on pigment fluorescence and photosynthetic capacity in Fremyella diplosiphon strains B481-WT and B481-SD was investigated. Our results indicated that both strains exposed to kanamycin from 0.2 to 3.2 mg/L and tetracycline from 0.8 to 12.8 mg/L enhanced growth and pigment accumulation. Additionally, B481-SD treated with 0.2-51.2 mg/L ampicillin resulted in a significant enhancement of pigment fluorescence. A detrimental effect on growth and pigmentation in both the strains exposed to 6.4-102.5 mg/L kanamycin and 0.8-102.5 mg/L cefotaxime was observed. Detection of reactive oxygen species revealed highest levels of oxidative stress at 51.2 and 102.5 mg/L kanamycin for B481-SD and 102.5 mg/L for B481-WT. Membrane permeability detected by lactate dehydrogenase assay indicated maximal activity at 0.8 mg/L ampicillin, kanamycin, and tetracycline treatments on day 6. Abundant vacuolation, pyrophosphate, and cyanophycin granule formation were observed in treated cells as a response to antibiotic stress. These findings on the hormetic effect of antibiotics on F. diplosiphon indicate that optimal antibiotic concentrations induce cellular growth while high concentrations severely impact cellular functionality. Future studies will be aimed to enhance cellular lipid productivity at optimal antibiotic concentrations to disintegrate the cell wall, thus paving the way for clean bioenergy applications.

Zero-Valent Iron Nanoparticles Induce Reactive Oxygen Species in the Cyanobacterium, Fremyella diplosiphon.

Nanoscale zero-valent iron nanoparticles (nZVIs) are known to boost biomass production and lipid yield in Fremyella diplosiphon, a model biodiesel-producing cyanobacterium. However, the impact of nZVI-induced reactive oxygen species (ROS) in F. diplosiphon has not been evaluated. In the present study, ROS in F. diplosiphon strains (B481-WT and B481-SD) generated in response to nZVI-induced oxidative stress were quantified and the enzymatic response determined. Lipid peroxidation as a measure of oxidative stress revealed significantly higher malondialdehyde content (p < 0.01) in both strains treated with 3.2, 12.8, and 51.2 mg L-1 nZVIs compared to untreated control. In addition, ROS in all nZVI-treated cultures treated with 1.6-25.6 mg L-1 nZVIs was significantly higher than the untreated control as determined by the 2',7'-dichlorodihydrofluorescein diacetate fluorometric probe. Immunodetection using densitometric analysis of iron superoxide dismutase (SOD) revealed significantly higher SOD levels in both strains treated with nZVIs at 51.2 mg L-1. In addition, we observed significantly higher (p < 0.001) SOD levels in the B481-SD strain treated with 6.4 mg L-1 nZVIs compared to 3.2 mg L-1 nZVIs. Validation using transmission electron microscopy equipped with energy-dispersive X-ray spectroscopy (EDS) revealed adsorption of nZVIs with a strong iron peak in both B481-WT and B481-SD strains. While the EDS spectra showed strong signals for iron at 4 and 12 days after treatment, a significant decrease in peak intensity was observed at 20 days. Future efforts will be aimed at studying transduction mechanisms that cause metabolic and epigenetic alterations in response to nZVIs in F. diplosiphon.

Enhancing the Performance of Dye Sensitized Solar Cells Using Silver Nanoparticles Modified Photoanode.

In this study, silver nanoparticles were synthesized, characterized, and applied to a dye-sensitized solar cell (DSSC) to enhance the efficiency of solar cells. The synthesized silver nanoparticles were characterized with UV-Vis spectroscopy, dynamic light scattering, transmission electron microscopy, and field emission scanning electron microscopy. The silver nanoparticles infused titanium dioxide film was also characterized by Fourier transform infrared and Raman spectroscopy. The performance of DSSC fabricated with silver nanoparticle-modified photoanode was compared with that of a control group. The current and voltage characteristics of the devices as well as the electrochemical impedance measurements were also carried out to assess the performance of the fabricated solar cells. The solar-to-electric efficiency of silver nanoparticles based DSSC was 1.76%, which is quite remarkable compared to the 0.98% realized for DSSC fabricated without silver nanoparticles.

Impact of Zero-Valent Iron Nanoparticles on Fremyella diplosiphon Transesterified Lipids and Fatty Acid Methyl Esters.

Efforts to enhance the transformative potential of biofuels is an important step to achieving an environment-friendly and sustainable energy source. Fremyella diplosiphon is an ideal third-generation biofuel agent due to its ability to produce lipids and desirable essential fatty acids. In this study, the impact of Nanofer 25s nanoscale zero-valent iron nanoparticles (nZVIs) on total lipid content and fatty acid composition of F. diplosiphon strains SF33 and B481 was investigated. We observed significant increases (P < 0.05) in the growth of F. diplosiphon treated with 0.2-1.6 mg L-1 Nanofer 25s, indicating that trace concentrations of nZVIs were not toxic to the organism. Chlorophyll a, carotenoids, and phycobiliprotein levels were not altered in F. diplosiphon treated with nZVIs ranging from 0.4 to 1.6 mg L-1, confirming that these concentrations did not negatively impact photosynthetic efficacy. In addition, Nanofer 25s ranging from 0.2 to 1.6 mg L-1 had an optimal impact on SF33 and B481 total lipid content. We identified significant increases in unsaturated fatty acid methyl esters (FAMEs) from F. diplosiphon Nanofer 25s-treated transesterified lipids. Theoretical chemical and physical biofuel properties revealed a product with elevated cetane number and oxidative stability for both strains. Scanning electron microscopy and energy-dispersive X-ray spectroscopy validated the localization of nZVIs. Our findings indicate that Nanofer 25s nZVIs significantly enhance F. diplosiphon total lipid content and essential FAMEs, thus offering a promising approach to augment the potential of the cyanobacterium as a large-scale biofuel agent.

Augmenting Fremyella diplosiphon Cellular Lipid Content and Unsaturated Fatty Acid Methyl Esters Via Sterol Desaturase Gene Overexpression.

Cyanobacteria have immense prospective as a platform for renewable energy; however, a major barrier in achieving optimal productivity is the low lipid yield. Fremyella diplosiphon, a model cyanobacterium, is an ideal biofuel agent due to its desirable fatty acid methyl esters (FAMEs). To enhance lipid content, we overexpressed the sterol desaturase (SD) gene in F. diplosiphon B481 wild type by genetic transformation. This effort resulted in a transformant (B481-SD) with a 64-fold increase in the SD gene at the mRNA transcript level, with no loss in growth and pigmentation. The transformant was persistently grown for over 32 generations indicating long-term stability and vitality. We observed 27.3% and 23% increases in total lipid content and unsaturated FAMEs respectively in B481-SD transesterified lipids with methyl octadecadienoate as the most abundant unsaturated component. In addition, we detected an 81% increase in FAME composition in the transformant compared with the wild type. Theoretical physical and chemical properties confirmed a FAME profile with very high cetane number (65.972-67.494) and oxidative stability (50.493-18.66 h) in the engineered strain. Results of the study offer a promising approach to augment F. diplosiphon total lipid content and unsaturated FAMEs, thus paving the way to enhance biofuel capacity of the organism.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

Microcystin Levels in Selected Cyanobacteria Exposed to Varying Salinity.

Microcystins produced by cyanobacteria pose a great threat to human health by releasing toxins upon cell death. In the present study, we studied microcystin production in the cyanobacterial strains Anabaena cylindrica (B629 and 2949) and Fremyella diplosiphon (SF33) exposed to 1, 2 and 4 g/L sodium chloride (NaCl). Cultures grown for 7 days in BG11/HEPES medium were pelleted, re-grown in the corresponding NaCl levels, and enzyme linked immunosorbent assay (ELISA) performed. ELISA assays revealed enhanced microcystin production in A. cylindrica B629 exposed to 4 g/L NaCl and A. cylindrica 29414 exposed to 2 and 4 g/L NaCl, after growth in the corresponding NaCl levels for 14 days. We observed a significant decrease (p >0.05) in microcystin levels in the control strains after exposure to NaCl for 5 days. After exposure to 1, 2, or 4 g/L NaCl for 10 days, no microcystin release was observed in A. cylindrica B629, A. cylindrica 29414 or F. diplosiphon SF33. Sodium dodecyl sulfate polyacrylamide gel electrophoresis identified the presence of an additional band at 120 - 130 kDa in A. cylindrica B629 exposed to 2 and 4 g/L NaCl, and at 14 kDa in cultures amended with 1 and 2 g/L NaCl as well as the untreated control, indicating that exposure to salinity induces alterations in protein expression.

Nanoparticle-mediated Impact on Growth and Fatty Acid Methyl Ester Composition in the Cyanobacterium Fremyella diplosiphon.

Insufficient light supply is a major limitation in cultivation of cyanobacteria for scaled up biofuel production and other biotechnological applications, which has driven interest in nanoparticle-mediated enhancement of cellular light capture. In the present study, Fremyella diplosiphon wild type (Fd33) and halotolerant (HSF33-2) strains were grown in solution with 20, 100, and 200 nm-diameter gold nanoparticles (AuNPs) to determine their impact on biomass accumulation, pigmentation, and fatty acid methyl ester (FAME) production. Results revealed a significant increase in growth of Fd33 (0.244 ± 0.006) and HSF33-2 (0.112 ± 0.003) when treated with 200 nm AuNPs. In addition, we observed a significant increase in chlorophyll a accumulation in 200 nm AuNP-treated Fd33 (25.7%) and HSF33-2 (36.3%) indicating that NPs enhanced photosynthetic pigmentation. We did not observe any alteration in FAME composition and biodiesel properties of transesterified F. diplosiphon lipids among all AuNP treatments. Interactions between F. diplosiphon and AuNPs were visualized using scanning electron microscopy. Energy dispersive X-ray spectroscopy confirmed the presence of AuNPs outside cells with aggregation in high cell density locales. Our findings indicate that nanotechnological approaches could significantly enhance growth of the organism with no negative effect on FAME-derived biodiesel properties, thus augmenting F. diplosiphon as a potential biofuel agent.

Fremyella diplosiphon as a biodiesel agent: Identification of fatty acid methyl esters via microwave-assisted direct in situ transesterification.

Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, Fremyella diplosiphon, was identified by fatty acid methyl esters (FAME) via direct transesterification. Total lipids in wild type (Fd33) and halotolerant (HSF33-1 and HSF33-2) strains determined by gravimetric analysis yielded 19% cellular dry weight (CDW) for HSF33-1 and 20% CDW for HSF33-2, which were comparable to Fd33 (18% CDW). Gas chromatography-mass spectrometry detected a high ratio of saturated to unsaturated FAMEs (2.48-2.61) in transesterified lipids, with methyl palmitate being the most abundant (C16:0). While theoretical biodiesel properties revealed high cetane number and oxidative stability, high cloud and pour point values indicated that fuel blending could be a viable approach. Significantly high FAME abundance in total transesterified lipids of HSF33-1 (40.2%) and HSF33-2 (69.9%) relative to Fd33 (25.4%) was identified using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry, indicating that robust salt stress response corresponds to higher levels of extractable FAME. Alkanes, a key component in conventional fuels, were present in F. diplosiphon transesterified lipids across all strains confirming that natural synthesis of these hydrocarbons is not inhibited during biodiesel production. While analysis of photosynthetic pigments and phycobiliproteins did not reveal significant differences, FAME abundance varied significantly in wild type and halotolerant strains indicating that photosynthetic pathways are not the sole factors that determine fatty acid production. We characterize the potential of F. diplosiphon for biofuel production with FAME yields in halotolerant strains higher than the wild type with no loss in photosynthetic pigmentation.

Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants.

Camelina sativa (C. sativa), an oilseed species rich in poly-unsaturated fatty acids, has gained great importance as an industrial oil platform crop in recent years. Despite the potential benefits of C. sativa for bioenergy applications, limited research has been conducted to improve its agronomic qualities. Hence, a simple and efficient technique for production of transgenic C. sativa plants is warranted. In the present study, shoot apical meristems of two C. sativa cultivars (Pl650159 and Pl650161) were transformed with Agrobacterium strain 'EHA 105' harboring the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase II (nptII) genes. After two days of co-cultivation in the dark, explants were transferred to selection medium. Transgenic shoots were identified on the basis of green fluorescence and kanamycin resistance. Shoots were then rooted and transferred to potting mix soil for acclimatization. This protocol describes an efficient method to generate transgenic C. sativa plants in as little as 4 weeks.

Overexpression of hlyB and mdh genes confers halotolerance in Fremyella diplosiphon, a freshwater cyanobacterium.

Fremyella diplosiphon is a freshwater cyanobacterium that has great potential as a biofuel agent due to its ability to grow in low light intensity and acclimation to different wavelengths. To enhance its halotolerance for growth in 35gL-1 sodium chloride (NaCl), plasmids harboring hemolysin B (hlyB) and malate dehydrogenase (mdh) genes were transformed into wild type F. diplosiphon (WT-Fd33). Electroporation-mediated overexpression of the genes resulted in two transformants, HSF33-1 and HSF33-2, with 9- and 20-fold increases in hlyB and mdh transcript levels. In addition, up-regulation of proteins at the expected size ranges of 50-60kDa for HlyB and 40-50kDa for MDH was observed. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed a protein spot corresponding to HlyB in HSF33-1 with a significant MOWSE score of 164 and 3% sequence coverage, and a spot corresponding to MDH in HSF33-2 gave a significant MOWSE score of 124 with 10% sequence coverage. Physiological evaluation in BG11/HEPES medium and seawater adjusted to 35gL-1 NaCl confirmed that the transformants could thrive in high salinity with no loss of photosynthetic pigments. Results of the study indicate that overexpression of hlyB and mdh genes confer halotolerance in F. diplosiphon, thus maximizing its potential as a large-scale biofuel agent.

Identification of a Halotolerant Mutant via In Vitro Mutagenesis in the Cyanobacterium Fremyella diplosiphon.

Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. In this study, the effect of salinity on the freshwater cyanobacterium, Fremyella diplosiphon, was investigated and mutagenesis-based efforts were undertaken to enhance salt tolerance. Salinity at a concentration of 10 g/L sodium chloride (NaCl) inhibited growth of wild type F. diplosiphon under white, red, and green light. Efforts to enhance halotolerance resulted in a mutant that could survive in 20 g/L NaCl for 15 generations with no significant reduction in phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) or chlorophyll a. Gene expression measured by quantitative reverse transcription-polymerase chain reaction revealed a three-fold increase in tripartite ATP-independent periplasmic transporters (TRAP) solute receptor transcript in the mutant compared to wild type. Our discovery of a TRAP transporter system in F. diplosiphon and its possible role in salinity response enables growth in brackish waters, which enhances its potential for biotechnological applications.