Marine Cyanobacterial Strains Research Articles

Development of an efficient process to extract C‐phycocyanin from marine cyanobacterial strains and evaluation of its bioactive potential

AbstractBACKGROUNDPhycocyanin (PC), a natural blue photosynthetic pigment found in cyanobacteria, rhodophyta and cryptophyta has numerous applications in biotechnology and the food industry. The phycocyanin extraction method has a significant impact on its bioactivities, purity index, cost and potential applications; thus, the development of an efficient extraction strategy continues to be a primary research goal.RESULTSThis study evaluated the efficacy of different methods for the extraction of cyanobacterial phycocyanin (C‐PC) from three marine cyanobacterial isolates, namely Arthrospira platensis, Phormidium sp. and Pseudoscillatoria sp. wet biomass. The pretreatment step of soaking biomass in extraction buffer for 60 min, followed by a combination of freeze–thaw (one cycle; 2 h) and sonication (2 min) treatment was found to be the most effective extraction strategy, producing the highest C‐PC yield (16.19 ± 0.34%, 15.89 ± 0.61% and 15.56 ± 0.69% dry cell weight for strains A. platensis, Phormidium sp. and Pseudoscillatoria sp., respectively). Furthermore, a biomass‐to‐solvent ratio of 0.02 g mL−1 was optimal to attain food‐grade purity ((A620/A280) ≥ 0.7). Bioactivity evaluation of crude and purified C‐PC exhibited its potential as antioxidant compounds and as an antimicrobial agent with significant inhibitory effects against various pathogenic microorganisms.CONCLUSIONThe study proposed an efficient and cost‐effective C‐PC extraction process for cyanobacteria and highlighted the potential of C‐PC as a valuable bioactive compound with various applications. © 2024 Society of Chemical Industry (SCI).

Cyclopropane-Containing Specialized Metabolites from the Marine Cyanobacterium cf. Lyngbya sp.

Marine cyanobacteria are known to produce structurally diverse bioactive specialized metabolites during bloom occurrence. These ecologically active allelochemicals confer chemical defense for the microalgae from competing microbes and herbivores. From a collection of a marine cyanobacterium, cf. Lyngbya sp., a small quantity of a new cyclopropane-containing molecule, benderadiene (2), and lyngbyoic acid (1) were purified and characterized using spectroscopic methods. Using live reporter quorum-sensing (QS) inhibitory assays, based on P. aeruginosa PAO1 lasB-gfp and rhlA-gfp strains, both compounds were found to inhibit QS-regulated gene expression in a dose-dependent manner. In addition to lyngbyoic acid being more active in the PAO1 lasB-gfp biosensor strain (IC50 of 20.4 µM), it displayed anti-biofilm activity when incubated with wild-type P. aeruginosa. The discovery of lyngbyoic acid in relatively high amounts provided insights into its ecological significance as a defensive allelochemical in targeting competing microbes through interference with their QS systems and starting material to produce other related analogs. Similar strategies could be adopted by other marine cyanobacterial strains where the high production of other lipid acids has been reported. Preliminary evidence is provided from the virtual molecular docking of these cyanobacterial free acids at the ligand-binding site of the P. aeruginosa LasR transcriptional protein.

Acclimation and Characterization of Marine Cyanobacterial Strains Euryhalinema and Desertifilum for C-Phycocyanin Production.

This study involves evaluation of two native cyanobacterial strains Euryhalinema and Desertifilum isolated from a mangrove pond in Haikou (China) for their possible phycocyanin (C-PC) production. Maximal growth rate with highest chlorophyll and C-PC accumulation were observed at 28°C and 60 μmol photons m−2 s−1 photon flux density for Euryhalinema sp., while for Desertifilum sp. at 32°C and 80 μmol photons m−2 s−1. Nitrogen and iron concentration trails revealed that double strength concentration of sodium nitrate and ferric ammonium citrate in original BG11 media increased growth rate and accumulation of C-PC for both strains. Three different C-PC extraction methods were tested. The combined extraction protocol of freeze–thaw and ultrasonication markedly increased the C-PC extraction efficiency and attained the food grade purity (A 620/A 280 ratio >0.7), whereas a higher C-PC yield was found with Na-phosphate buffer. Furthermore, the clarified crude extract was used to purify C-PC by fractional ammonium sulfate [(NH₄)₂SO₄] precipitation, Sephadex G-25 gel filtration chromatography, and DEAE-sephadex ion exchange chromatography and attained analytical grade purity (A 620/A 280 ratio >3.9). Taken together, both strains showed their potential to be domesticated for valuable phycocyanin production.

Changes in Population Age-Structure Obscure the Temperature-Size Rule in Marine Cyanobacteria.

The temperature-size Rule (TSR) states that there is a negative relationship between ambient temperature and body size. This rule has been independently evaluated for different phases of the life cycle in multicellular eukaryotes, but mostly for the average population in unicellular organisms. We acclimated two model marine cyanobacterial strains (Prochlorococcus marinus MIT9301 and Synechococcus sp. RS9907) to a gradient of temperatures and measured the changes in population age-structure and cell size along their division cycle. Both strains displayed temperature-dependent diel changes in cell size, and as a result, the relationship between temperature and average cell size varied along the day. We computed the mean cell size of new-born cells in order to test the prediction of the TSR on a single-growth stage. Our work reconciles previous inconsistent results when testing the TSR on unicellular organisms, and shows that when a single-growth stage is considered the predicted negative response to temperature is revealed.

Novel marine cyanobacteria from the Atlantic coast of Brazil

ABSTRACT Marine cyanobacteria have emerged as a source of promising bioactive compounds. The isolation of strains from tropical environments seems to be an important step in the search for molecules displaying bioactivity, besides being valuable for biotechnological purposes. Thus, the objective of the present study was to describe marine cyanobacterial strains, isolated and characterized in terms of morphology and molecular phylogeny, as well as screened for biologically active compounds. Five strains were isolated and morphologically characterized as Leptolyngbya (CENA553, CENA554 and CENA555) and Geitlerinema (CENA552 and CENA556). Considering the phylogenetic results, these strains did not match the clusters of the type-species or reference strains, indicating the possible emergence of new generic entities morphologically related to Leptolyngbya and Geitlerinema. Among the isolated strains, CENA552 and CENA556 were selected for chemical analyses. Five known nucleosides with biological activities and two amino acids were recognized and isolated from the CENA556 strain. The GC-MS analyses of strains CENA552 and CENA556 revealed distinct classes of non-polar compounds, with a predominance of octadecane (CENA556), 2-hexyl-1-decanol (CENA552) and neophytadiene (CENA552 and CENA556) hydrocarbons. All nucleosides and amino acids were highly abundant, indicating these morphotypes as promising sources of these bioactive compounds. This is the first report considering taxonomy/molecular phylogeny as well as chemistry prospection of morphotypes resembling Geitlerinema-like isolated from the Brazilian coastal area, highlighting the potential of these taxa for biotechnological applications.

Glycogen Production in Marine Cyanobacterial Strain Synechococcus sp. NKBG 15041c.

An important feature offered by marine cyanobacterial strains over freshwater strains is the capacity to grow in seawater, replacing the need for often-limited freshwater. However, there are only limited numbers of marine cyanobacteria that are available for genetic manipulation and bioprocess applications. The marine unicellular cyanobacteria Synechococcus sp. strain NKBG 15041c (NKBG15041c) has been extensively studied. Recombinant DNA technologies are available for this strain, and its genomic information has been elucidated. However, an investigation of carbohydrate production, such as glycogen production, would provide information for inevitable biofuel-related compound production, but it has not been conducted. In this study, glycogen production by marine cyanobacterium NKBG15041c was investigated under different cultivation conditions. NKBG15041c yielded up to 399μg/ml/OD730 when cells were cultivated for 168h in nitrogen-depleted medium (marine BG11ΔN) after medium replacement (336h after inoculation). Cultivation under nitrogen-limited conditions also yielded an accumulation of glycogen in NKBG15041c cells (1mM NaNO3, 301μg/ml/OD730; 3mM NaNO3, 393μg/ml/OD730; and 5mM NaNO3, 328μg/ml/OD730) under ambient conditions. Transcriptional analyses were carried out for 13 putative genes responsible for glycogen synthesis and catabolism that were predicted based on homology analyses with Synechocystis sp. PCC 6803 (PCC6803) and Synechococcus sp. PCC7002 (PCC7002). The transcriptional analyses revealed that glycogen production in NKBG15041c under nitrogen-depleted conditions can be explained by the contribution of both increased carbon flux towards glycogen synthesis, similar to PCC6803 and PCC7002, and increased transcriptional levels of genes responsible for glycogen synthesis, which is different from the conventionally reported phenomenon, resulting in a relatively high amount of glycogen under ambient conditions compared to PCC6803 and PCC7002.

Inhibitory effect of marine cyanobacterial extract on biofilm formation and virulence factor production of bacterial pathogens causing vibriosis in aquaculture

Emergence of extended antibiotic resistance among bacterial pathogens often leads to the failure of existing antibiotics to treat bacterial infections; therefore, there is an urgent need to look for novel alternative treatment measures. The aim of this study was to evaluate the anti-quorum sensing (QS) potential of Synechococcus sp., to prevent the onset of bacterial infections as an alternate to antibiotics. A total of 110 marine cyanobacterial strains were screened for their anti-QS activity against biomarker strain Chromobacterium violaceum (ATCC 12472) and aquatic bacterial pathogens Vibrio harveyi (MTCC 3438) and Vibrio vulnificus (MTCC 1145). Of the 110 strains tested, the extract of unicellular algae Synechococcus sp. (Q-25) exhibited the efficient reduction in the production of violacein pigment of C. violaceum to the level of 82 %, bioluminescence of V. harveyi to 91 % and protease in V. vulnificus to 63 %. In V. harveyi and V. vulnificus, it exhibited a significant reduction of 71 and 84 % in biofilm formation and 66 and 68 % in EPS production, respectively, without any antibacterial activity. Confocal laser scanning microscopic and light microscopic analyses further confirmed that the Q-25 extract effectively prevented initial attachment as well as disrupting the architecture of mature biofilm, when compared to their untreated controls. In addition, the characterization of active principle by gas chromatography–mass spectrometry analysis confirmed the presence of stable bioactive compound hexadecanoic acid in the extract. Hence, this study clearly revealed the antibiofilm and QS inhibitory potential of the cyanobacterium, Synechococcus sp.

The development and characterization of an exogenous green-light-regulated gene expression system in marine cyanobacteria.

A green-light-regulated gene expression system derived from Synechocystis sp. PCC 6803 was constructed and introduced into the marine cyanobacterial strain Synechococcus sp. NKBG 15041c. The regulation system was evaluated using gfp uv as a reporter gene under red-light illumination and under simultaneous red- and green-light illumination. Expression of the reporter gene was effectively repressed under red-light illumination and increased over 10-fold by illuminating with green light. Control vectors missing either the ccaS sensor histidine kinase gene or the ccaR response regulator gene showed no detectable induction of GFPuv expression. Green-light induction of gfp uv expression was further confirmed by quantitative reverse transcription PCR. The constructed system was effective at regulating the recombinant expression of a target gene using green light in a marine cyanobacterial strain that does not naturally possess such a green-light regulation system. Thus, constructed green-light-regulated gene expression system may be used as a core platform technology for the development of marine cyanobacterial strains in which bioprocesses will be regulated by light.

Enhanced photobiological H2 production by the addition of carbon monoxide and hydrogen cyanide in two unicellular N2-fixing cyanobacterial strains isolated from Korean coasts

Photobiological H2 from marine cyanobacterial strains is widely accepted to be an ideal clean and renewable energy source. Using the two Korean N2-fixing unicellular cyanobacterial strains (Cyanothece sp. KNU CB MAL-031 and Cyanothece sp. KNU CB MAL-058) and the Synechococcus sp. Miami strain BG043511 we performed flask-scale experiments to measure the effect of CO and HCN addition on photobiological H2 production. For the test, 1, 5, 10 and 30% v/v of CO in the N2 atmosphere was applied. Enhancement of H2 production was remarkable at 1–5% concentration range of CO addition. At CO concentrations over 5% no further cost-effective enhancement of H2 production was detectable, which suggests to us that 1–5% CO addition should be adopted for practical photobiological H2 production by the cyanobacterial strains. Maximum enhancement of the photobiological H2 production by CO additions was 2–6 times over the control flasks without CO. When 3 ppm of HCN was injected into the cell suspension of BG043511, the enhancement of hydrogen production was 50–60% of that under 5% CO. Present result implies the possible recycling of waste CO and HCN for the enhancement of the photobiological H2 production using marine cyanobacterial strains.

Improved Protein Overexpression and Purification Strategies for Structural Studies of Cyanobacterial Metal-Responsive Transcription Factor, SmtB from Marine Synechococcus sp. PCC 7002

There are structural and functional differences in SmtB homologs, metal-responsive transcription factors responsible for sensing of excess heavy metal ions in marine and freshwater cyanobacterial strains. The structure of SmtB from freshwater Synechococcus sp. strain PCC 7942 is elucidated with nuclear magnetic resonance (NMR) and crystallography techniques. But knowledge about the functioning of SmtB homologs from marine species is limited till date. To enable NMR spectroscopic studies for investigating structural and functional aspects, modified protocols with higher yields of isotopically labeled SmtB, from marine species like Synechococcus sp. PCC 7002 are essential. In this study, smtB gene was cloned from genome of Synechococcus sp. PCC 7002 and overexpression protocol for recombinant SmtB was standardized in Escherichia coli containing T7 RNA polymerase/promoter system. Further, the protocol for large-scale production, isotope labeling with (15)N, and purification of recombinant SmtB in E. coli BL21(DE3)/pLysS cells was developed. Purified recombinant protein was successfully used for NMR spectroscopy experiments. These results indicate that the overexpression technique now developed is applicable to the structural and functional studies for the proteins being homologous to cyanobacterial SmtB from Synechococcus sp. PCC 7002.

Phylogenetic Inferences Reveal a Large Extent of Novel Biodiversity in Chemically Rich Tropical Marine Cyanobacteria

Benthic marine cyanobacteria are known for their prolific biosynthetic capacities to produce structurally diverse secondary metabolites with biomedical application and their ability to form cyanobacterial harmful algal blooms. In an effort to provide taxonomic clarity to better guide future natural product drug discovery investigations and harmful algal bloom monitoring, this study investigated the taxonomy of tropical and subtropical natural product-producing marine cyanobacteria on the basis of their evolutionary relatedness. Our phylogenetic inferences of marine cyanobacterial strains responsible for over 100 bioactive secondary metabolites revealed an uneven taxonomic distribution, with a few groups being responsible for the vast majority of these molecules. Our data also suggest a high degree of novel biodiversity among natural product-producing strains that was previously overlooked by traditional morphology-based taxonomic approaches. This unrecognized biodiversity is primarily due to a lack of proper classification systems since the taxonomy of tropical and subtropical, benthic marine cyanobacteria has only recently been analyzed by phylogenetic methods. This evolutionary study provides a framework for a more robust classification system to better understand the taxonomy of tropical and subtropical marine cyanobacteria and the distribution of natural products in marine cyanobacteria.

Marine Benthic Cyanobacteria Contain Apoptosis-Inducing Activity Synergizing with Daunorubicin to Kill Leukemia Cells, but not Cardiomyocytes

The potential of marine benthic cyanobacteria as a source of anticancer drug candidates was assessed in a screen for induction of cell death (apoptosis) in acute myeloid leukemia (AML) cells. Of the 41 marine cyanobacterial strains screened, more than half contained cell death-inducing activity. Several strains contained activity against AML cells, but not against non-malignant cells like hepatocytes and cardiomyoblasts. The apoptotic cell death induced by the various strains could be distinguished by the role of caspase activation and sensitivity to the recently detected chemotherapy-resistance-associated prosurvival protein LEDGF/p75. One strain (M44) was particularly promising since its activity counteracted the protective effect of LEDGF/p75 overexpressed in AML cells, acted synergistically with the anthracycline anticancer drug daunorubicin in AML cells, and protected cardiomyoblasts against the toxic effect of anthracyclines. We conclude that culturable benthic marine cyanobacteria from temperate environments provide a promising and hitherto underexploited source for novel antileukemic drugs.

M13-based genotyping of marine cyanobacterial strains from the Indian subcontinent and maintained in the NFMC germplasm collection

The aim of this study was to analyze marine cyanobacterial culture collections strains of the Indian subcontinent at the level below species. This is important to improve the abilities of service culture collections to provide their user community with correctly identified and clean organisms. A total of 50 marine cyanobacterial strains were genotyped with M13 polymerase chain reaction (PCR) fingerprinting to provide diagnostic fingerprints for each culture. Depending on the strains, 9 to 26 bands were observed for the primer tested. Within the species, strains representing different isolates were genetically clearly different. Data obtained from genomic fingerprinting were used to construct binary distance matrix, and the neighbor-joining tree constructed demonstrated the ability of this method to differentiate strains at the intraspecific level. An important and useful result obtained in this study is the application of the M13 PCR fingerprinting method on almost all forms of cyanobacteria for strain and species discrimination.

Laccase and polyphenol oxidase activities of marine cyanobacteria: a study with Poly R-478 decolourization

The various marine cyanobacterial strains tested showed wide variation in growth patterns and decolourization patterns of the lignin model polymeric dye Poly R-478. The study revealed the presence of laccases (LACs) and polyphenol oxidases (PPOs) in marine cyanobacteria. All the ten tested strains were found to possess constitutive PPOs, whereas only four strains showed the presence of constitutive laccases. Within 7 days of incubation the highest percentage of decolourization was shown by Phormidium valderianum BDU140441 (65%), and Oscillatoria chlorina BDU 140691 (12%) showed the least. Isoforms of LACs were found to be induced by the laccase elicitors veratryl aldehyde, caffeic acid, guaiacol and tannic acid. Cyanobacterial strains that possess both LACs and PPOs were relatively more efficient in decolourizing the dye. Altering the concentrations of nitrogen, phosphorus, potassium and sulphur from the basal medium influenced the efficiency of dye decolourization.

Toxicity assessment of Indian marine cyanobacterial strains.

Toxicity assessment of Indian marine cyanobacterial strains.

Structure, function and regulation of the cyanobacterial high-affinity bicarbonate transporter, BCT1.

The cmpABCD operon of the cyanobacterium Synechococcus sp. strain PCC7942, which is transcribed specifically under CO2-limited growth conditions, encodes an ATP-binding cassette (ABC) transporter involved in HCO3-uptake (designated BCT1). The product of the cmpA gene is a 42-kDa protein anchored to the plasma membrane, which binds HCO3- with high affinity (Kd= 5 µM) and acts as the substrate-binding protein of the transporter. The apparent Km(HCO3-) of BCT1 is 15 µM. BCT1 has the highest affinity for HCO3- among the HCO3- transporters of the Synechococcus strain and is essential for competitive utilization of HCO3- under CO2-limited conditions. BCT1 is closely related to the cyanobacterial nitrate/nitrite transporter (NRT) encoded by the nrtABCD genes. The BCT1 and NRT transporters, together with the putative cyanate transporter of cyanobacteria, comprise a monoanion transporter subfamily in the family of ABC importers. BCT1 and NRT are present in most fresh-water strains of cyanobacteria but seem to be absent in marine cyanobacterial strains. The low CO2-responsive induction of thecmp operon requires a LysR family protein CmpR, which is similar to CbbR (RbcR), the activator of the CO2 fixation operons of chemoautotrophic and purple photosynthetic bacteria.