Externally supplied ascorbic acid moderates detrimental effects of UV-C exposure in cyanobacteria.

The defensive role performed by exogenously supplied ascorbic acid in the cyanobacterium Nostoc muscorum Meg1 against damages produced by UV-C radiation exposure was assessed in this study. Exposure to UV-C (24 mJ/cm2) significantly enhanced reactive oxygen species (ROS) (50%) along with peroxidation of lipids (21%) and protein oxidation (22%) in the organism. But, addition of 0.5 mM ascorbic acid prior to UV-C exposure showed reduction in ROS production (1.7%) and damages to lipids and proteins (1.5 and 2%, respectively). Light and transmission electron microscopic studies revealed that ascorbic acid not only protected filament breakage but also restricted severe ultrastructural changes and cellular damages in the organism. Although the growth of the organism was repressed up to 9% under UV-C treatment within 15 days, a pre-treatment with ascorbic acid led to growth enhancement by 42% in the same period. Various growth parameters such as photo-absorbing pigments (phycoerythrin, phycocyanin, allophycocyanin, chlorophyll a, and carotenoids), water splitting complex (WSC), D1 protein, RuBisCO, glutamine synthetase and nitrogenase activities in the UV-C treated organism were seen to be relatively intact in the presence of ascorbic acid. Thus, a detailed analysis undertaken in the present study was able to demonstrate that ascorbic acid not only act as first responder against harmful UV-C radiation by down-regulating ROS production, it also accelerated the growth performance in the organism in the post UV-C incubation period as an immediate response to an adverse experience presented in the form of UV-C radiation exposure.

Cyanobacterial degradation of herbicide 2,4-dichlorophenoxyacetic acid (2,4-D): Its response to the oxidative stress induced by the primary degradation product 2,4-dichlorophenol (2,4-DCP).

Excessive use of herbicides in agricultural fields has become a major environmental concern due to the negative effects on the ecosystem. Microbial degradation has been well-known as an effective approach for combating such non-natural substances in soil. In the present study, the degradation of 2,4-Dichlorophenoxyacetic acid (2,4-D) as a result of metabolic activities of a cyanobacterium Nostoc muscorum Meg 1 was investigated using GC-MS analysis. After seven days of 2,4-D exposure, the main residue obtained was 2,4-dichlorophenol (2,4-DCP) at RT: 8.334 (confirmed using NIST library). The effects of 2,4-DCP were studied in a cyanobacterium Nostoc muscorum Meg 1 isolated from a rice field where 2,4-D is commonly used. Exposure to 2,4-DCP at 20, 40, and 80 ppm significantly increased ROS production in the cyanobacterium by 74, 107, and 211 % (p < 0.001). With rising 2,4-DCP concentrations in the surroundings, lipid peroxidation and protein oxidation in the organism correspondingly increased, indicating cellular injury. The mRNA and protein contents, and also the activities of different oxidant neutralizing enzymes such as CAT, SOD, GR, and GPx and the non-enzymatic antioxidants (proline, GSH, thiol and phytochelatin content) were found augmented in 20 ppm 2,4-DCP exposed cultures. However, in the presence of 40 and 80 ppm 2,4-DCP, most enzymatic and non-enzymatic antioxidants were severely compromised. At higher exposures, the organism's attempt to mitigate the oxidants was still visible, as both proline and TSH levels increased. SEM and TEM analysis aided in visualizing the effects of 2,4-DCP on the morphology and ultrastructures of the organism.

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.

Modulation in isocitrate dehydrogenase activity under citrate enrichment affects carbon and nitrogen fixations in the cyanobacterium Nostoc muscorum Meg 1.

The enzyme isocitrate dehydrogenase (IDH) converts isocitrate synthesized from citrate to α-ketoglutarate in the TCA cycle. In cyanobacteria, α-KG has an additional role where it donates its carbon skeleton for ammonium assimilation in the GS-GOGAT pathway thereby linking carbon and nitrogen metabolisms. Looking at this crucial function of IDH that makes α-KG available for both carbon and nitrogen assimilation, changes brought about in its activity under excess availability of citrate in a cyanobacterium was evaluated. Further, how these changes are transmitted downstream affecting carbon and nitrogen metabolisms were also evaluated. A 100 µM citrate supplementation induced IDH activity. Consequently, there was an increase in concentrations of photosynthetic pigments, D1 protein and RuBisCO as well as in PSII activity. Heterocyst differentiation was initiated and an upsurge in the activities of nitrogenase and GS were recorded. An enhancement in the total protein and carbohydrate content reiterated the positive influence of citrate enrichment on carbon and nitrogen fixation. The increase in the mRNA contents of IDH, D1 protein, RuBisCO, nitrogenase and GS indicated their induction at the genetic level. Finally, there was augmentation in total biomass production by ∼28%. Interestingly as citrate concentration was increased to 500 µM, both C- and N- fixations were highly compromised suggesting that even though citrate is an essential metabolite in the cells, it became toxic beyond a certain concentration to the organism. SEM and TEM studies showed no changes in the organism's morphology and ultra-structure in presence of 100 µM citrate while adverse changes were noticed in presence of 500 µM citrate.

Analyzing dose dependency of antioxidant defense system in the cyanobacterium Nostoc muscorum Meg 1 chronically exposed to Cd2.

The aim of the present study was to analyze the dose dependency of oxidant-antioxidant homeostasis in Cd2+ exposed Nostoc muscorum Meg 1 cells. Quantification of percent DNA loss, protein oxidation and lipid peroxidation was carried out to assess Cd2+ induced ROS mediated damages to the organism. The countermeasures adopted by the cyanobacterium were also evaluated by computing various components of both enzymatic and non-enzymatic antioxidants. Exposure to different Cd2+ (0.1, 0.2, 0.3, 0.5, 1, 1.5, 2, 2.5, 3 ppm) doses showed substantial increase in ROS content in the ranges of 20-181% and 116-323% at the end of first and seventh day. The DNA damage, protein oxidation and lipid peroxidation were increased by 11-62%, 7-143% and 13-183% with increasing Cd2+ concentrations at the end of seven days. TEM images clearly showed damages to the cell wall, cell membrane and thylakoid organization at higher Cd2+ (0.5-3 ppm) concentrations. Cd2+ exposure up to 0.5 ppm registered increase in contents of antioxidative enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)) and in non-enzymatic antioxidants (glutathione, total thiol, phytochelatin and proline) indicating stimulation of ROS mitigating machinery. However, toxicity of Cd2+ was evident as at higher concentrations the cellular morphology and ultra-structures were negatively affected and the capacities of the cells to generate various antioxidant measures were highly compromised. The organism registered 96-98% sorption ability from a solution supplemented with 0.3 ppm Cd2+ and thus show realistic potential as Cd2+ bioremediator in wastewater treatment.

Unstandardized UV-C dose used for killing harmful cyanobacteria may instead initiate accelerated growth in the target organisms.

Although UV-C radiation has been in use for killing unwanted cyanobacteria, experiments with lower doses of UV-C radiation instead showed induction of growth related parameters and enhanced biomass production in the cyanobacterium Nostoc muscorum Meg1. When the cyanobacterial cultures were exposed to UV-C radiation of varying doses (6, 12 and 18 mJ/cm2), concentrations of various photo-absorbing pigments, RuBisCO and D1 protein of PSII; activities of oxygen evolving complex, nitrogenase and glutamine synthetase were significantly increased upon 6 and 12 mJ/cm2 UV-C radiation exposures. Resulting higher photosynthetic performance was evident from the augmentation in carbohydrate content by ∼49% under single exposure to 6 mJ/cm2 UV-C by fifteenth day. The increased performances of both RuBisCO and D1 proteins were in part also due to induction at the genetic level as seen from the increase in their mRNA and protein levels under treatment. Similar increase was also observed in protein (16%) and in lipid contents (43%) that reflected an upsurge in the total biomass. Highest biomass (463 mg/L/d) was noted in culture exposed to 6 mJ/cm2 UV-C radiation, representing a ∼25% increase. Furthermore the possibility of this organism using part of the incident UV-C radiation as an additional source of energy was deduced from an experiment where the thylakoid membranes excited within UV (226-400 nm) range showed emission at longer wavelengths with an emission maximum at ∼640 nm. Thus this work provides evidence that lower UV-C doses can potentially augment cyanobacterial growth and use of unstandardized UV-C doses for restricting cyanobacterial growth may in fact produce contrary result.

Modulation of oxidant and antioxidant homeostasis in the cyanobacterium Nostoc muscorum Meg1 under UV-C radiation stress.

The present work was conducted to study how restoration of perturbed oxidant and antioxidant homeostasis is achieved in the UV-C radiation exposed cells of cyanobacterium Nostoc muscorum Meg1. Exposure to varying doses of UV-C radiation (6, 12, 18 and 24 mJ/cm2) showed damage to ultrastructures especially cytoplasmic membrane, cell wall and organisation of thylakoid membranes of the cyanobacterium under transmission electron microscope (TEM). All doses of UV-C exposure significantly induced most of the enzymatic antioxidant {catalase, superoxide dismutase (SOD) and glutathione reductase (GR)} activities, their protein levels (western blot analysis) and mRNA levels (real time PCR analysis) within the first hour of post UV-C radiation incubation period. In the same way, contents of many non-enzymatic antioxidants such as ascorbic acid, reduced glutathione, proline, phenol and flavonoids were also augmented in response to such UV-C radiation exposure. Although notable increase in ROS level was only seen in cultures treated with 24 mJ/cm2 UV-C exposure which also registered increase in protein oxidation (22%) and lipid peroxidation (20%), this boost in both enzymatic and non-enzymatic antioxidants was significant in all radiation exposed cells indicating cell's preparation to combat rise in oxidants. Further, albeit all antioxidants increased considerably, their levels were restored back to control values by day seventh re-establishing physiological redox state for normal metabolic function. The combined efficiency of the enzymatic and non-enzymatic antioxidants were so effective that they were able to bring down the increase levels of ROS, lipid peroxidation and protein oxidation to the physiological levels within 1 h of radiation exposure signifying their importance in the defensive roles in protecting the organism from oxidative toxicity induced by UV-C radiation exposure.

Dose dependent variance in UV-C radiation induced effects on carbon and nitrogen metabolism in the cyanobacterium Nostoc muscorum Meg1.

With the intention of getting an insight into the differential effect of UV-C radiation on the N2-fixing heterocystous cyanobacterium Nostoc muscorum Meg1, various aspects of carbon and nitrogen metabolism was evaluated in the organism. Exposure to different doses of UV-C (6, 12, 18 and 24 mJ/cm2) showed that among various photo-absorbing pigments, phycobiliproteins were most sensitive. Oxygen evolving complex (OEC) activity measured as net oxygen evolution rate decreased by 63% upon 24 mJ/cm2 exposure. Western blot analysis established that D1 protein of PSII was highly sensitive and its levels decreased even at a radiation dose as low as 6 mJ/cm2. In contrast, levels of the Calvin cycle enzyme RuBisCO was increased at 6 and 12 mJ/cm2 doses but the level decreased drastically (84%) at higher dose (24 mJ/cm2). The nitrogenase enzyme activity decreased at all doses but the ammonia assimilating enzyme glutamine synthetase (GS) activity recorded increase at the lower doses. The reactive oxygen species (ROS) and lipid peroxidation increased upon UV-C exposure. Transmission electron microscopic observation revealed damage to ultrastructure especially the thylakoid membrane organization, aggregation of dissolving phycobilisomes and loss of caboxysomes. Interestingly, sub-lethal radiation (6 and 12 mJ/cm2) dose exposures increased the growth rate in the organism when growth was measured over a period of 11 days after radiation exposure.

Zn2+ sequestration by Nostoc muscorum: study of thermodynamics, equilibrium isotherms, and biosorption parameters for the metal.

Microbial biosorption has evolved as an effective strategy for heavy metal removal from contaminated waters. The common cyanobacterium Nostoc muscorum isolated from the banks of a polluted river in Meghalaya, India, was tested for its potential to remove Zn2+ from aqueous solutions. Energy-dispersive X-ray (EDX) study verified Zn binding on the cyanobacterial biomass, and FTIR analysis revealed many negatively charged functional groups (hydroxyl, carbonyl, alcohol, amine, phosphoryl, sulfhydryl, and carboxyl) on the cell surface that aided in metal binding. Thermodynamic studies established the biosorption process to be energetically favorable with negative free energy change (-10.404, -10.599, and -10.796 kJ/mol at 298, 303, and 308 K, respectively). Sorption isotherm data fitted best in the Langmuir isotherm indicating monolayer nature of Zn sorption. The organism showed hyper-accumulation tendency towards Zn with a maximum sorption capacity as high as 2500 mg of Zn taken up per gram of biomass. The separation factor R L calculated from Langmuir isotherm ranged between 0 and 1 signifying favorable interaction between the cyanobacterial biomass and the Zn ions. Various experimental parameters, viz. pH, temperature, inoculum age and size, and shaking rate, influenced Zn biosorption. Optimized experimental conditions significantly enhanced the sorption percentage. Sorption was primarily a fast surface phenomenon in the beginning with internalization of zinc ions by the live cells on prolonged exposure.

Biosorption and equilibrium isotherms study of cadmium removal by Nostoc muscorum Meg 1: morphological, physiological and biochemical alterations.

Rice fields of Meghalaya especially in the coal mining belt receive water contaminated by effluents from mines that are known to carry harmful heavy metal ions such as Cu, Fe, Zn, Ni, Cd, As, Pb, Cr, etc. Cd exposure was analyzed in the cyanobacterium Nostoc muscorum Meg 1 isolated from a contaminated rice field in Sohra, Meghalaya, India. Toxicity study established 0.5 ppm on day 3 to be the LD50. At LD50 chlorophyll a and total protein concentration was reduced by 50.9 and 52.5%, while nitrogenase and glutamine synthetase activities were inhibited by 40.8 and 38.4%. EDX and FTIR analyses confirmed Cd binding and participation of hydroxyl, carbonyl, carboxyl and phosphate groups in biosorption of Cd onto the cell surfaces. SEM study established morphological changes. At pH 8.0 and temperature 25 ± 2 °C, the cyanobacterium removed 92% Cd within 24 h. Of this, 91% Cd was adsorbed on the cell surface while 4% was internally accumulated. The energy required for internal accumulation of Cd was partly provided in the form of ATP synthesized during active photosynthesis. The Langmuir isotherm was found best fitted with a R 2 value 0.98 when compared to Freundlich and Temkin adsorption isotherms. The maximum sorption capacity, Q max, of the organism was 71.4 mg of Cd per g of biomass. R L value of 0.29 indicated favorable interaction between cyanobacterial biomass and Cd. The adsorption intensity, n value 7.69 g/L obtained from Freundlich isotherm showed that the organism possessed high Cd sorption capacity.

Heavy metal removal from multicomponent system by the cyanobacterium Nostoc muscorum: kinetics and interaction study.

In this study, Nostoc muscorum, a native cyanobacterial species isolated from a coal mining site, was employed to remove Cu(II), Zn(II), Pb(II) and Cd(II) from aqueous solution containing these metals in the mixture. In this multicomponent study, carried out as per the statistically valid Plackett-Burman design of experiments, the results revealed a maximum removal of both Pb(II) (96.3 %) and Cu(II) (96.42 %) followed by Cd(II) (80.04 %) and Zn(II) (71.3 %) at the end of the 60-h culture period. Further, the removal of these metals was attributed to both passive biosorption and accumulation by the actively growing N. muscorum biomass. Besides, the specific removal rate of these metals by N. muscorum was negatively correlated to its specific growth rate. For a better understanding of the effect of these metals on each other's removal by the cyanobacteria, the results were statistically analyzed in the form of analysis of variance (ANOVA) and Student's t test. ANOVA of the metal bioremoval revealed that the main (individual) effect due to the metals was highly significant (P value <0.05) on each other's removal. Student's t test results revealed that both Zn(II) and Pb(II) strongly inhibited both Cu(II) removal (P value <0.01) and Cd(II) removal (P value <0.02). All these results not only demonstrated a very good potential of the cyanobacteria in the bioremoval of these metals but also the effect of individual metals on each other's removal in the multicomponent system.

Zn(II) and Cu(II) removal by Nostoc muscorum: a cyanobacterium isolated from a coal mining pit in Chiehruphi, Meghalaya, India.

Nostoc muscorum was isolated from a coal mining pit in Chiehruphi, Meghalaya, India, and its potential to remove Zn(II) and Cu(II) from media and the various biochemical alterations it undergoes during metal stress were studied. Metal uptake measured as a function of the ions removed by N. muscorum from media supplemented independently with 20 µmol/L ZnSO4 and CuSO4 established the ability of this cyanobacterium to remove 66% of Zn(2+) and 71% of Cu(2+) within 24 h of contact time. Metal binding on the cell surface was found to be the primary mode of uptake, followed by internalization. Within 7 days of contact, Zn(2+) and Cu(2+) mediated dissimilar effects on the organism. For instance, although chlorophyll a synthesis was increased by 12% in Zn(2+)-treated cells, it was reduced by 26% in Cu(2+)-treated cells. Total protein content remained unaltered in Zn(2+)-supplemented medium; however, a 15% reduction was noticed upon Cu(2+) exposure. Copper enhanced both photosynthesis and respiration by 15% and 19%, respectively; in contrast, photosynthesis was unchanged and respiration dropped by 11% upon Zn(2+) treatment. Inoculum age also influenced metal removal ability. Experiments in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosynthetic inhibitor), carbonyl cyanide m-chlorophenyl hydrazone (an uncoupler), and exogenous ATP established that metal uptake was energy dependent, and photosynthesis contributed significantly towards the energy pool required to mediate metal removals.

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum.

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues L-methionine-DL-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in 35S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.

Isolation and characterization of a Mastigocladus species capable of growth, N(2)-fixation and N-assimilation at elevated temperature.

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2-3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2-3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO(3) (-) and NH(4) (+), respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO(3) (-)-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH(4) (+))-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.