Nitric oxide mediates positive regulation of Nostoc flagelliforme polysaccharide yield via potential S-nitrosylation of G6PDH and UGDH.

Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni+-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.

A newly discovered glycosyltransferase gene UGT88A1 affects growth and polysaccharide synthesis of Grifola frondosa.

Grifola frodosa polysaccharides, especially ß-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. KEY POINTS: •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.

Salicylic Acid and Jasmonic Acid Increase the Polysaccharide Production of Nostoc flagelliforme via the Regulation of the Intracellular NO Level.

To significantly improve the polysaccharide production of Nostoc flagelliforme, a total of 12 chemicals were evaluated for their effects on polysaccharide accumulation. The results showed that salicylic acid and jasmonic acid increased the accumulation of the polysaccharides in N. flagelliforme significantly, by more than 20%. Three polysaccharides, namely control-capsule polysaccharide, salicylic acid-capsule polysaccharide, and jasmonic acid-capsule polysaccharide, were extracted and purified from N. flagelliforme under normal, salicylic acid, and jasmonic acid culture conditions, respectively. Their chemical compositions slightly differed regarding the total sugar and uronic acid contents, with average molecular weights of 2.06 × 103, 2.16 × 103 and 2.04 × 103 kDa, respectively. They presented similar Fourier transform infrared spectra and no significant difference in antioxidant activity. It was revealed that the salicylic acid and jasmonic acid significantly increased the level of nitric oxide. By investigating the effects of the exogenous nitric oxide scavenger and nitric oxide donor on the nitric oxide levels and polysaccharide yield of N. flagelliforme, the results showed that the increase in intracellular nitric oxide levels might be an important factor promoting the accumulation of polysaccharides. These findings provide a theoretical foundation for enhancing the yield of secondary metabolites by regulating the intracellular nitric oxide levels.

Four polysaccharides isolated from Poria cocos mycelium and fermentation broth supernatant possess different activities on regulating immune response.

Four polysaccharide fractions were isolated and purified from the culture supernatant and mycelium of Poria cocos, and differences in their immunomodulatory activity were investigated. The average molecular weights of EPS-0M, EPS-0.1M, IPS-0M, and IPS-0.1M were 1.77 × 103, 2.01 × 103, 0.03 × 103 and 4.97 × 103 kDa, respectively. They all mainly consisted of 5 monosaccharides, including glucose, mannose, galactose, fucose and rhamnose, but with different molar ratios. At a dose of 50 µg/mL, EPS-0M, EPS-0.1M, and IPS-0.1M significantly increased the production of nitric oxide (NO), as well as the mRNA and protein levels of pro-inflammatory factors including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW264.7 cells, suggesting that they enhanced macrophage-mediated innate immunity. Moreover, based on the in vitro inflammation model of lipopolysaccharide (LPS)-stimulated RAW264.7 cells, EPS-0M, EPS-0.1M and IPS-0M but not IPS-0.1M could inhibit the LPS-induced excessive inflammatory response, including NO, IL-6, TNF-α, IL-1ß production and gene transcription. Interestingly, IPS-0M showed a relatively poor immunostimulatory effect, but had the strongest inhibitory effect against the LPS-induced RAW264.7 inflammatory response. Furthermore, our results indicate that the nuclear factor-kappa B (NF-κB) pathway is associated with the immunomodulatory effects of the polysaccharide samples on RAW264.7 cells. This study can provide a reference for the more targeted application of different polysaccharide components from Poria cocos for human health.

Transcription level differences in Taxus wallichiana var. mairei elicited by Ce3+, Ce4+ and methyl jasmonate.

Taxol is a precious and effective anticancer drug. Cerium and methyl jasmonate (MJ) have been shown to increase the yield of taxol in taxus cells. However, the mechanisms of cerium-mediated and MJ-mediated taxol biosynthesis remain unknown. RNA-Seq was applied to study the overall regulation mechanism of cerium and MJ on taxol biosynthesis and analyze the differences among T. mairei cells elicited by Ce3+, Ce4+ and MJ on transcriptional level . Using sequence homology, 179 unigenes were identified as taxol synthesis genes. Under the condition of 100 µM MJ, taxol synthesis genes were up-regulated. Notably, taxol synthesis genes were down-regulated expression at 1 mM Ce3+ and 1 mM Ce4+. Differential expression genes involved in some related functions were analyzed, such as MAPK signaling pathway and plant-pathogen interaction. Sequence alignment and phylogenetic analysis of nine differentially expressed WRKYs in our data were carried out.

[Autophagy Activation and Mitochondrial Quality Control in Sarcopenia].

Sarcopenia,an age-related disease caused by the imbalance in protein synthesis and degradation,can result in significant decreases in skeletal muscle mass and strength.Skeletal muscle loss during aging is inevitable and can affect the life quality of the elderly.Moreover,it may increase the risks of other age-related diseases in the elderly.However,the underlying molecular mechanism remains unclear in age-related skeletal muscle loss.Autophagy is a degradation pathway for the removal of dysfunctional organelles and damaged macromolecules during aging.Mitochondria also play a key role in skeletal muscle function.To maintain skeletal muscle mass,we should pay attention to autophagy and improve mitochondrial homeostasis through autophagy or other means.This paper summarizes the research progress of autophagy and mitochondrial quality control in sarcopenia,aiming to provide reference for exploring new therapies.

Nostoc flagelliforme capsular polysaccharides from different culture conditions improve hyperlipidemia and regulate intestinal flora in C57BL/6J mice to varying degrees.

Two capsular polysaccharides (WL-CPS-1 and GLU-CPS-1) purified from Nostoc flagelliforme under normal and mixotrophic culture conditions were used to investigate the hypolipidemic activity and effect on intestinal flora in C57BL/6J mice respectively. Their molecular weight and monosaccharide composition have been determined in previous studies. They both improved the lipid level by affecting the expression of lipid metabolism genes. They down-regulated the TNF-α and IL-1ß levels in serum and up-regulated the activity of antioxidant enzymes in liver thus decreased the atherosclerosis index and MDA content. They up-regulated the short chain fatty acids (SCFAs) synthesis. They decreased the abundance of pathogenic bacteria and increased the abundance of probiotics positively correlated with SCFAs. Compared with WL-CPS-1, GLU-CPS-1 exhibited higher in vivo activity and enriched Odoribacter and Alloprevotella correlating with the gene expression of lipid metabolism, suggesting that the bioactivity of polysaccharides could be regulated by culture conditions. These findings contributed to application of N. flagelliforme polysaccharides with higher activity in hypolipidemia by adjusting culture conditions.

The effects of quorum sensing molecule farnesol on the yield and activity of extracellular polysaccharide from Grifola frondosa in liquid fermentation.

A strategy by exogenous addition of quorum sensing molecule farnesol to improve the production, antioxidant activity and antitumor activity of extracellular polysaccharide (EPS) of Grifola frondosa by liquid fermentation was proposed in the study. The highest yield of EPS induced by farnesol was 1.25 g/L, which was 150% higher than that of the control. Four polysaccharides including EPS-C-0M, EPS-C-0.2M, EPS-F-0M and EPS-F-0.2M were extracted and purified under the conditions of control and farnesol respectively. The physicochemical properties, antioxidant activities and antitumor activities were studied. Their chemical composition differed in sugar, protein and uronic acid contents, and they were composed of six constituent monosaccharides with different ratios, with the average molecular weights of 1.12 × 103, 1.89 × 103, 1.41 × 103 and 2.02 × 103 kDa, respectively. They presented similar FT-IR spectra, but different surface morphology. Antioxidant experiments showed that they had strong scavenging activities on ABTS+, hydroxyl radical, O2- and DPPH radical. Antitumor experiments showed that they had strong inhibitory effects on human cervical cancer (HeLa) cells and human liver cancer cells (HepG2) cells. Among the four polysaccharides, EPS-F-0.2M showed the highest antioxidant and antitumor activities, indicating that farnesol could regulate the biological activity of EPS by affecting structure and properties. These results demonstrated that appropriate adjustment of culture conditions had potential application in the development of polysaccharides with high antioxidant and antitumor activity. It provided a new strategy to enhance the production and bioactivity of edible and medicinal fungal polysaccharides by using quorum sensing molecules.

Improvement of Biomineralization of Sporosarcina pasteurii as Biocementing Material for Concrete Repair by Atmospheric and Room Temperature Plasma Mutagenesis and Response Surface Methodology.

Microbially induced calcium carbonate precipitation (MICP) has recently become an intelligent and environmentally friendly method for repairing cracks in concrete. To improve on this ability of microbial materials concrete repair, we applied random mutagenesis and optimization of mineralization conditions to improve the quantity and crystal form of microbially precipitated calcium carbonate. Sporosarcina pasteurii ATCC 11859 was used as the starting strain to obtain the mutant with high urease activity by atmospheric and room temperature plasma (ARTP) mutagenesis. Next, we investigated the optimal biomineralization conditions and precipitation crystal form using Plackett-Burman experimental design and response surface methodology (RSM). Biomineralization with 0.73 mol/l calcium chloride, 45 g/l urea, reaction temperature of 45°C, and reaction time of 22 h, significantly increased the amount of precipitated calcium carbonate, which was deposited in the form of calcite crystals. Finally, the repair of concrete using the optimized biomineralization process was evaluated. A comparison of water absorption and adhesion of concrete specimens before and after repairs showed that concrete cracks and surface defects could be efficiently repaired. This study provides a new method to engineer biocementing material for concrete repair.

Clinical observation for acupuncture treatment of a small area of hyperplastic scars in young and middle-aged women.

INTRODUCTION: Hypertrophic scars are a common disease in plastic surgery, which is the reaction of skin connective tissue to trauma beyond the normal range. Although scholars around the world have explored the tissue structure and formation mechanism of HS for decades, they are not satisfactory the result of. No effective treatment has been found. Therefore, the search for safe and effective treatments for HS has always been the focus of medical attention and research. Acupuncture therapy has a definite effect on HS and has unique advantages. METHODS/DESIGN: In this study, we will use our own front-to-back clinical research method. We plan to include 120 young and middle-aged female patients who meet the diagnostic criteria for HS. The untreated HS of the enrolled patients will be used as blank controls. The intervention group will be given acupuncture treatment. The assessment of scar area, color, hardness, thickness, itching and pain will be recorded for 30 days of treatment. DISCUSSION: This trial may provide evidence regarding the clinical effectiveness, safety, and cost-effectiveness of Acupuncture for patients with HS. TRIAL REGISTRATION: ClinicalTrials.gov, ChiCTR2000032624, Registered on 04 May 2020.

A comparative study on the clinical effect of the treatment of pathological scars in young and middle-aged women with the combination of Chinese and western medicine.

INTRODUCTION: Pathological scar is the abnormal manifestation of skin fiber hyperplasia caused by the failure of normal healing after skin damage. At present, there are many clinical treatments for pathological scars. However, there is no cure for clinically effective pathological scars with high recurrence rate. In this study, we will use a combination of Chinese and western medicine treatment methods to evaluate the clinical efficacy and related indicators of young and middle-aged female patients who meet pathological scars, looking for an objective and effective treatment method for pathological scars. METHODS/DESIGN: In this study, we will use our own front-to-back clinical research method. We plan to include 120 young and middle-aged female patients who meet the diagnostic criteria for pathological scars. The untreated pathological scars of the enrolled patients will be used as blank controls. The intervention group will be given conventional western medicine treatment and combined Chinese and western medicine treatment. The assessment of scar area, color, hardness, thickness, itching, and pain was recorded for 8 weeks of treatment. DISCUSSION: This trial may provide evidence regarding the clinical effectiveness, safety, and cost-effectiveness of traditional Chinese medicine for patients with pathological scars. TRIAL REGISTRATION: ClinicalTrials.gov, ChiCTR2000032187, Registered on April 22, 2020.

Comparisons of Functional Properties of Polysaccharides from Nostoc flagelliforme under Three Culture Conditions.

Nostoc flagelliforme is an edible cyanobacterium with excellent food and herbal values. It has been used as food in China for more than 2000 years. Many studies have been focused on improving the yield and bioactivity of Nostoc flagelliforme polysaccharides although these have ignored the functional properties. In this study, we extracted and purified three polysaccharides (WL-CPS, NaCl-CPS and Glu-CPS) from Nostoc flagelliforme under normal, salt stress and mixotrophic culture conditions, respectively, in order to change the physicochemical properties of polysaccharides with the aim of obtaining better functional properties. Both salt stress and mixotrophic culture conditions increased the specific yield of polysaccharides. Their functional properties were comparatively investigated and the results showed that NaCl-CPS exhibited the highest emulsification activity and flocculation capability, which was also higher than that of some commercial products. In contrast, Glu-CPS exhibited the highest water and oil holding capacities, foaming property, intrinsic viscosity and bile acids binding capacity. Our results indicated that both NaCl-CPS and Glu-CPS could be considered to be functional polysaccharides according to their respective characteristics, which have great potential in numerous applications, such as food, pharmaceutical, cosmetic, chemical and mineral industries. These findings also demonstrated the potential application of the proper regulation of culture conditions in the development of polysaccharides with desired functional properties.

ROS Is a Factor Regulating the Increased Polysaccharide Production by Light Quality in the Edible Cyanobacterium Nostoc flagelliforme.

To explore the regulatory factor of light quality affecting exopolysaccharide (EPS) production, transcriptome analysis of Nostoc flagelliforme cells exposed to red light (R), blue light (B), and mixed light (B/R = 15:7) (BR) with white fluorescent light as control was performed. The differentially expressed genes mainly enriched in carbohydrate metabolism and energy metabolism. Significant enrichment in the oxidation-reduction process and energy metabolism indicated that intracellular redox homeostasis was disrupted. An assay of reactive oxygen species (ROS) and malondialdehyde contents demonstrated light quality induced oxidative stress. To illustrate the relationship between ROS level and EPS accumulation, the effects of the exogenous addition of ROS scavenger N-acetyl cysteine and inducer H2O2 on the oxidation-reduction level and EPS production were compared. The results revealed that light quality regulated EPS biosynthesis via the intracellular ROS level directly other than oxidative stress. Understanding such relationships might provide guidance for efficient EPS production to regulate the intracellular redox level.

Comparative proteomic analysis of Nostoc flagelliforme reveals the difference in adaptive mechanism in response to different ultraviolet-B radiation treatments.

Nostoc flagelliforme is a pioneer organism in the desert and highly resistant to ultraviolet B (UV-B) radiation, while the involved adaptive mechanism has not been fully explored yet. To elucidate the responsive mechanism, two doses of UV-B radiation (low: 1 W/m2 and high: 5 W/m2) were irradiated for 6 h and 48 h, respectively, and their effects on global metabolism in N. flagelliforme were comprehensively investigated. In this study, we used iTRAQ-based proteomic approach to explore the proteomes of N. flagelliforme, and 151, 172, 124 and 148 differentially expressed proteins were identified under low and high UV-B doses for 6 h and 48 h, respectively. Functional classification analysis showed these proteins were mainly involved in photosynthesis, amino acid metabolism, antioxidant activity and carbohydrate metabolism. Further analysis revealed that UV-B imposed restrictions on primary metabolism including photosynthesis, Calvin cycle, and amino acid metabolism, and cells started defense mechanism through repair of DNA and protein damage, increasing antioxidant activity, and accumulating extracellular polysaccharides to minimize the damage. Moreover, high UV-B dose imposed more severe restrictions and activated stronger defense mechanism compared with low dose. The results would improve the understanding of molecular mechanisms of UV-B-stress adaption in N. flagelliforme.

Less toxic zinc(ii), diorganotin(iv), gallium(iii) and cadmium(ii) complexes derived from 2-benzoylpyridine N,N-dimethylthiosemicarbazone: synthesis, crystal structures, cytotoxicity and investigations of mechanisms of action.

Four metal complexes based on 2-benzoylpyridine N,N-dimethylthiosemicarbazone (Bp44mT) were designed. Free ligand and zinc(ii), diorganotin(iv), gallium(iii) and cadmium(ii) complexes all demonstrated pronounced activity, which was indicated using the growth inhibition test in vitro. Interestingly, most of the compounds were found to be selective against hepatocellular carcinoma (HepG2) cells but had little effect on normal hepatocyte (QSG7701) cells. In particular, Zn(Bp44mT)2 (1) exhibited toxicity on QSG7701 cells which approximately 12-fold lower than that on HepG2 cells. The studies of mechanisms of action indicated that 1 induced reactive oxygen species (ROS) generation in a dose-dependent manner via the mitochondria transduction pathway. Protein analyses showed that 1 significantly promoted p21 and p53 gene expression, causing caspase-3 activation.

Effect of culture conditions on the physicochemical properties and antioxidant activities of polysaccharides from Nostoc flagelliforme.

Three polysaccharides (WL-CPS-1, NaCl-CPS-1 and Glu-CPS-1) were extracted and purified from Nostoc flagelliforme under normal, salt stress and mixotrophic culture conditions respectively. Their physicochemical properties and antioxidant activities were investigated. WL-CPS-1, NaCl-CPS-1 and Glu-CPS-1 chemical composition differed in sugar and uronic acid contents, and they were composed of nine constituent monosaccharides and one uronic acid with different ratios, with the average molecular weights of 1.02 × 103, 1.12 × 103 and 1.33 × 103 kDa, respectively. They presented similar fourier transform infrared spectra, but different surface morphology, chain length and branching. Antioxidant assay showed that they all exhibited strong scavenging activity on ABTS+ and hydroxyl radicals and moderate activity on DPPH radical. Glu-CPS-1 exhibited the highest antioxidant activity suggested culture conditions could regulate the bioactivity through influencing the structure and properties. These findings demonstrated the potential application of proper regulation of culture conditions in the development of polysaccharides with high antioxidant activity.

The physiological responses of terrestrial cyanobacterium Nostoc flagelliforme to different intensities of ultraviolet-B radiation.

Nostoc flagelliforme is a pioneer organism in the desert and exerts important ecological functions. The habitats of N. flagelliforme are characterized by intense solar radiation, while the ultraviolet B (UV-B) tolerance has not been fully explored yet. To evaluate the physiological responses of N. flagelliforme to UV-B radiation, three intensities (1 W m-2, 3 W m-2 and 5 W m-2) were used, and the changes in photosynthetic pigments, cell morphology, mycosporine-like amino acids (MAAs) synthesis and cell metabolism were comparatively investigated. Under high UV-B intensity or long term radiation, chlorophyll a, allophycocyanin and phycocyanin were greatly decreased; scanning electron microscope observations showed that cell morphology significantly changed. To reduce the damage, cells synthesized a large amount of carotenoid. Moreover, three kinds of MAAs were identified, and their concentrations varied with the changes of UV-B intensity. Under 1 W m-2 radiation, cells synthesized shinorine and porphyra-334 against UV-B, while with the increase of intensity, more shinorine turned into asterine-330. Metabolite profiling revealed the contents of some cytoprotective metabolites were greatly increased under 5 W m-2 radiation. The principal component analysis showed cells exposed to UV-B were metabolically distinct from the control sample, and the influence on metabolism was particularly dependent on intensity. The results would improve the understanding of physiological responses of N. flagelliforme to UV-B radiation and provide an important theoretical basis for applying this organism to control desertification.

The relationship between monosaccharide composition of extracellular polysaccharide and activities of related enzymes in Nostoc flagelliforme under different culture conditions.

The relationship between monosaccharide composition of Nostoc flagelliforme extracellular polysaccharide (EPS) and activities of EPS synthesis enzymes under various carbon sources, nitrogen sources and light culture condition was investigated. Culture conditions showed significant influences on both monosaccharide composition and related enzyme activities. Under both carbon and nitrogen sources conditions, mannose mole percentage was increased with the increase of initial mole ratio of C/N and positively related to fructose-1, 6-bisphosphatase activity, and glucuronic acid and galactose mole percentages were positively correlated with UDP-glucose dehydrogenase, while arabinose and rhamnose mole percentages were negatively associated with UDP-glucose pyrophosphorylase. Different correlation between monosaccharide composition and enzymes activity from carbon and nitrogen sources conditions was found under light condition. These findings will be helpful to establish a novel fermentation process aimed to produce the N. flagelliforme EPS with desired monosaccharide composition.

Comparison of bacterial community structures of terrestrial cyanobacterium Nostoc flagelliforme in three different regions of China using PCR-DGGE analysis.

Filamentous Nostoc flagelliforme form colloidal complex, with beaded cells interacting with other bacteria embedded in the complex multilayer sheath. However, the species of bacteria in the sheath and the interaction between N. flagelliforme and associated bacteria remain unclear. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of N. flagelliforme from three regions of China. DGGE patterns showed variations in all samples, exhibiting 25 discrete bands with various intensities. The diversity index analysis of bands profiles suggested the high similarity of bacterial communities to each other but also the dependence of microbial composition on each location. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Cyanobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, of which Cyanobacteria was dominant, followed the Proteobacteria. Members of the genus Nostoc were the most abundant in all samples. Rhizobiales and Actinobacteria were identified, whereas, Craurococcus, Caulobacter, Pseudomonas, Terriglobus and Mucilaginibacter were also identified at low levels. Through comparing the bacterial composition of N. flagelliforme from different regions, it was revealed that N. flagelliforme could facilitate the growth of other microorganisms including both autotrophic bacteria and heterotrophic ones and positively contributed to their harsh ecosystems. The results indicated N. flagelliforme played an important role in diversifying the microbial community composition and had potential application in soil desertification.

Metabolomic approach to optimizing and evaluating antibiotic treatment in the axenic culture of cyanobacterium Nostoc flagelliforme.

The application of antibiotic treatment with assistance of metabolomic approach in axenic isolation of cyanobacterium Nostoc flagelliforme was investigated. Seven antibiotics were tested at 1-100 mg L(-1), and order of tolerance of N. flagelliforme cells was obtained as kanamycin > ampicillin, tetracycline > chloromycetin, gentamicin > spectinomycin > streptomycin. Four antibiotics were selected based on differences in antibiotic sensitivity of N. flagelliforme and associated bacteria, and their effects on N. flagelliforme cells including the changes of metabolic activity with antibiotics and the metabolic recovery after removal were assessed by a metabolomic approach based on gas chromatography-mass spectrometry combined with multivariate analysis. The results showed that antibiotic treatment had affected cell metabolism as antibiotics treated cells were metabolically distinct from control cells, but the metabolic activity would be recovered via eliminating antibiotics and the sequence of metabolic recovery time needed was spectinomycin, gentamicin > ampicillin > kanamycin. The procedures of antibiotic treatment have been accordingly optimized as a consecutive treatment starting with spectinomycin, then gentamicin, ampicillin and lastly kanamycin, and proved to be highly effective in eliminating the bacteria as examined by agar plating method and light microscope examination. Our work presented a strategy to obtain axenic culture of N. flagelliforme and provided a method for evaluating and optimizing cyanobacteria purification process through diagnosing target species cellular state.