Label-Free Analysis and Sorting of Microalgae and Cyanobacteria in Microdroplets by Intrinsic Chlorophyll Fluorescence for the Identification of Fast Growing Strains.

Microalgae and cyanobacteria are promising organisms for sustainable biofuel production, but several challenges remain to make this economically viable, including identification of optimized strains with high biomass productivity. Here we report on a novel methodology for the label-free screening and sorting of cyanobacteria and microalgae in a microdroplet platform. We show for the first time that chlorophyll fluorescence can be used to measure differences in biomass between populations of picoliter microdroplets containing different species of cyanobacteria, Synechocystis PCC 6803 and Synechococcus PCC 7002, which exhibit different growth dynamics in bulk culture. The potential and robustness of this label-free screening approach is further demonstrated by the screening and sorting of cells of the green alga Chlamydomonas reinhardtii encapsulated in droplets.

Screening and genetic characterization of thermo-tolerant Synechocystis sp. PCC6803 strains created by adaptive evolution.

BACKGROUND: Temperature tolerance is an important aspect for commercial scale outdoor cultivation of microalgae and cyanobacteria. While various genes are known to be related to Synechocystis sp. PCC6803's heat shock response, there is very limited published data concerning the specific genes involved in long term thermal tolerance. We have previously used random mutagenesis and adaptive evolution to generate a mixture of strains of Synechocystis sp. PCC6803 with significantly increased thermal tolerance. The genetic modifications leading to the phenotypes of the newly generated strains are the focus of this work. RESULTS: We used a custom screening platform, based on 96-deepwell microplate culturing in an in house designed cultivation chamber integrated in a liquid handling robot for screening and selection; in addition we also used a more conventional system. The increased thermal tolerances of the isolated monoclonal strains were validated in larger bioreactors and their whole genomes sequenced. Comparison of the sequence information to the parental wild type identified various mutations responsible for the enhanced phenotypes. Among the affected genes identified are clpC, pnp, pyk2, sigF, nlpD, pyrR, pilJ and cya1. CONCLUSIONS: The applied methods (random mutagenesis, in vivo selection, screening, validation, whole genome sequencing) were successfully applied to identify various mutations, some of which are very unlikely to have been identified by other approaches. Several of the identified mutations are found in various strains and (due to their distribution) are likely to have occurred independently. This, coupled with the relatively low number of affected genes underscores the significance of these specific mutations to convey thermal tolerance in Synechocystis.

Metal removal of cyanobacterial exopolysaccharides by uronic acid content and monosaccharide composition.

In the present study, chromium, cadmium and metal mixed (chromium+cadmium) removal and its association with exopolysaccharides and uronic acids production in Synechocystis sp. BASO671 were investigated. It was investigated that BASO671 showed different removal ability when exposed to each metal solely and mixed metal. EPS production by BASO671 was increased following exposure to 15 and 35 ppm Cr(VI), Cd(II) and Cr(VI)+Cd(II). Monomer composition of EPS was changed after metal treatment. Uronic acid contents of metal treated cells were higher than control cells of each isolate. Also, glucuronic acid content and galactronic acid content of EPS correlated with uronic acid contents of cells. Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis confirmed that a considerable amount of metals had precipitated on the cell surface. Fourier transform infrared spectrum analysis of EPSs indicated the presence of CH and CO group, which may serve as binding sites for divalent cations.

Phenylalanine ammonia lyase (PAL) enzyme activity and antioxidant properties of some cyanobacteria isolates.

In the present study, six cyanobacteria isolates were evaluated for the PAL enzyme activity, and their methanol extracts were assessed for the total phenolic amount and other antioxidant parameters. Synechocystis sp. BASO444 and Synechocystis sp. BASO673 isolates with high levels of total phenols (66.0±1.2µg/mg, 78.1±1.8µg/mg, respectively) also showed high levels of PAL activities (20.5±3.1U/mg protein, 17.2±2.3U/mg protein, respectively) and strong antioxidant activities. To understand the effect of l-phenylalanine (l-phe) on the PAL activity, total phenolic amount, and phenolic constituents, isolates were evaluated with 100mg/l l-phe. While PAL activities exhibited no significant change with l-phe addition, total phenolic amount of the isolates significantly increased. HPLC analysis revealed gallic acid, trans-cinnamic acid, p-coumaric acid, and ferulic acid as the main compounds. Results suggested that the two isolate mights be an important source for the l-phe inducible phenolic compounds.

Statistical evaluation of nutritional components impacting phycocyanin production in Synechocystis SP

Alkaliphilic cyanobacterial cultures were isolated from Lonar lake (MS, India). Among the set of cultures, Synechocystis sp, was studied for phycocyanin production. A maximum yield was obtained in BG-11 medium at optimized conditions (pH 10 and 16 h light). In order to increase the phycocyanin yield media optimization based on the eight media components a Plackett-Burman design of the 12 experimental trials was used. As per the analysis CaCl2.2H2O and Na2CO3 have been found to be the most influencing media components at 95% significance. Further the optimum concentrations of these components were estimated following a Box Wilson Central Composite Design (CCD) with four star points and five replicates at the center points for each of two factors was adopted for optimization of these two media components. The results indicated that there was an interlinked influence of CaCl2.2H2O and Na2CO3 on 98% significance. The maximum yield of phycocyanin (12% of dry wt) could be obtained at 0.058 g/l and 0.115 g/l of CaCl2.2H2O and Na2CO3, respectively.

Separation of bacteria with imprinted polymeric films.

Separation of compounds out of complex mixtures is a key issue that has been solved for small molecules by chromatography. However, general methods for the separation of large bio-particles, such as cells, are still challenging. We demonstrate integration of imprinted polymeric films (IPF) into a microfluidic chip, which preferentially capture cells matching an imprint template, and separate strains of cyanobacteria with 80-90% efficiency, despite a minimal difference in morphology and fluorescence, demonstrating its general nature. It is currently thought that the imprinting process, conducted while the polymer cures, transfers chemical information of the cell's external structure to the substrate. Capture specificity and separation can be further enhanced by orienting the imprints parallel to the flow vector and tuning the pH to a lower range.

Destruction of Raman biosignatures by ionising radiation and the implications for life detection on Mars.

Raman spectroscopy has proven to be a very effective approach for the detection of microorganisms colonising hostile environments on Earth. The ExoMars rover, due for launch in 2018, will carry a Raman laser spectrometer to analyse samples of the martian subsurface collected by the probe's 2-m drill in a search for similar biosignatures. The martian surface is unprotected from the flux of cosmic rays, an ionising radiation field that will degrade organic molecules and so diminish and distort the detectable Raman signature of potential martian microbial life. This study employs Raman spectroscopy to analyse samples of two model organisms, the cyanobacterium Synechocystis sp. PCC 6803 and the extremely radiation resistant polyextremophile Deinococcus radiodurans, that have been exposed to increasing doses of ionising radiation. The three most prominent peaks in the Raman spectra are from cellular carotenoids: deinoxanthin in D. radiodurans and ß-carotene in Synechocystis. The degradative effect of ionising radiation is clearly seen, with significant diminishment of carotenoid spectral peak heights after 15 kGy and complete erasure of Raman biosignatures by 150 kGy of ionising radiation. The Raman signal of carotenoid in D. radiodurans diminishes more rapidly than that of Synechocystis, believed to be due to deinoxanthin acting as a superior scavenger of radiolytically produced reactive oxygen species, and so being destroyed more quickly than the less efficient antioxidant ß-carotene. This study highlights the necessity for further experimental work on the manner and rate of degradation of Raman biosignatures by ionising radiation, as this is of prime importance for the successful detection of microbial life in the martian near subsurface.

Degradation of cyanobacterial biosignatures by ionizing radiation.

Primitive photosynthetic microorganisms, either dormant or dead, may remain today on the martian surface, akin to terrestrial cyanobacteria surviving endolithically in martian analog sites on Earth such as the Antarctic Dry Valleys and the Atacama Desert. Potential markers of martian photoautotrophs include the red edge of chlorophyll reflectance spectra or fluorescence emission from systems of light-harvesting pigments. Such biosignatures, however, would be modified and degraded by long-term exposure to ionizing radiation from the unshielded cosmic ray flux onto the martian surface. In this initial study into this issue, three analytical techniques--absorbance, reflectance, and fluorescence spectroscopy--were employed to determine the progression of the radiolytic destruction of cyanobacteria. The pattern of signal loss for chlorophyll reflection and fluorescence from several biomolecules is characterized and quantified after increasing exposures to ionizing gamma radiation. This allows estimation of the degradation rates of cyanobacterial biosignatures on the martian surface and the identification of promising detectable fluorescent break-down products.

Detection and identification of potentially toxic cyanobacteria in Polish water bodies.

The main goal of this study was to determine the distribution of potentially toxic cyanobacteria in 39 selected Polish water bodies. From the water bodies with blooms and also from those in which blooms were not visible 87 samples were investigated. For the first time samples from ponds localized in villages with high agricultural activities were included. Lakes for which microcystin concentrations had been determined before were included as a reference for the research. The detection of cyanobacteria was conducted by microscopic observation as well as by PCR amplification of the rpoC1 gene fragment. Cyanobacteria were present in 75 out of 87 samples. The presence of potentially toxic cyanobacteria was detected by amplification of the mcyB and mcyE genes, which are involved in the biosynthesis of microcystins. Both genes were detected in 7 out of 9 blooms investigated. In the case of samples collected from water bodies in which blooms were not observed, the mcyB and mcyE genes were detected in 20 out of 36. In order to identify the cyanobacteria occurring in selected reservoirs, 16S plus ITS clone libraries were constructed. The method allowed distinguishing 18 different genotypes. After sequence analysis, cyanobacteria belonging to genera Microcystis, Planktothrix, Anabaena, Pseudanabaena, Synechocystis, Synechococcus and Woronichinia were identified. Results confirmed the usefulness of the rpoC1 and mcy genes for monitoring water bodies and detection of potentially toxic cyanobacteria. Application of molecular markers allowed detecting potentially toxic cyanobacteria before the bloom was visible. This is the first comprehensive study concerning cyanobacteria present in different types of Polish water bodies performed using molecular markers.

Cadmium(II) sequestration characteristics by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition.

We investigated cadmium(II) resistance and its association with exopolysaccharide (EPS) production in cyanobacteria. Increased EPS production was associated with Cd(II) resistance. The most resistant isolate, Synechocystis sp. BASO670, secreted the greatest amount of EPS (548 mg/L). EPS production by Synechocystis sp. BASO670 and Synechocystis sp. BASO672 was increased following exposure to 15 and 35 ppm Cd(II). Monomer composition of EPS belonging to each isolate was changed after Cd(II) treatment. Uronic acid contents of Cd(II) treated cells were higher than control cells of each isolate. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis confirmed that a considerable amount of metals had precipitated on the cell surface. Fourier transform infrared (FT-IR) spectrum analysis of EPSs belonging to both isolates indicated the presence of C-H and C-O group, which may serve as binding sites for divalent cations.

Rapid determination of cytokinins and auxin in cyanobacteria.

Five cyanobacterial strains, Anabaena sp. Ck1, Oscillatoria sp. Ck2, Phormidium sp. Ck3, Chroococcidiopsis sp. Ck4, and Synechosystis sp. Ck5 were selected for their positive cytokinins-like activity using cucumber cotyledon bioassay and GUS assay in Arabidopsis ARR5::GUS. Classical cucumber cotyledon bioassay was modified for direct screening of cyanobacteria avoiding need for extraction and purification. Cytokinins from cyanobacteria were absorbed onto filter paper which was then assayed for cytokinins-like activity. A rapid chromatographic method was developed for the simultaneous determination of cytokinins and indole-3-acetic acid (IAA). Cyanobacterial biomass (50-100 mg) and cell-free culture filtrate were extracted in Bieleski buffer and purified by solid-phase extraction. The extract was used to determine phytohormones by ultra performance liquid chromatography and electrospray ionization-tandem mass spectrometry in positive and negative modes, respectively, with multiple reactions monitoring. Stable isotope-labeled cytokinins and IAA standards were added in the samples to follow recovery of the compounds and method validation. Five cytokinins determined in the selected strains were Zeatin (cis and trans isomers), Zeatin riboside, Dihydrozeatin riboside, and zeatin-o-glucoside. The strains were shown to accumulate as well as release the phytohormones.

Uptake of Pb(2+ )by a cyanobacterium belonging to the genus Synechocystis, isolated from East Kolkata Wetlands.

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 mug ml(-1) Pb(2+ )and growth was completely inhibited at 400 mug ml(-1) Pb(2+). All experiments were carried out with 100 mug ml(-1) Pb(2+) in which growth was the maximum. 91.67% of the total Pb(2+) got adsorbed to the outer surface of the cell and 1% of the total Pb(2+) entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb(2+) were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR-SmtB like regulator gene was expressed in both the strains after Pb(2+) induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb(2+) induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb(2+).

Screening of cyanobacterial species for calcification.

Species of cyanobacteria in the genera Synechococcus and Synechocystis are known to be the catalysts of a phenomenon called "whitings", which is the formation and precipitation of fine-grained CaCO3 particles. Whitings occur when the cyanobacteria fix atmospheric CO2 through the formation of CaCO3 on their cell surfaces, which leads to precipitation to the ocean floor and subsequent entombment in mud. Whitings represent one potential mechanism for CO2 sequestration. Research was performed to determine the ability of various strains of Synechocystis and Synechococcus to calcify when grown in microcosms amended with 2.5 mM HCO(3-) and 3.4 mM Ca2+. Results indicated that although all strains tested have the ability to calcify, only two Synechococcus species, strains PCC 8806 and PCC 8807, were able to calcify to the extent that a CaCO3 precipitate was formed. Enumeration of the cyanobacterial cultures during testing indicated that cell density did not appear to have a direct effect on calcification. Factors that had the greatest effect on calcification were CO2 removal and subsequent generation of alkaline pH. Whereas cell density was similar for all strains tested, differences in maximum pH were demonstrated. As CO2 was removed, growth medium pH increased and soluble Ca2+ was removed from solution. The largest increases in growth medium pH occurred when CO2 levels dropped below 400 ppmv. Research presented demonstrates that, under the conditions tested, many species of cyanobacteria in the genera Synechocystis and Synechococcus are able to calcify but only two species of Synechococcus were able to calcify to an extent that led to the precipitation of calcium carbonate.