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Abstract Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2% biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi protected Synechocystis against the development of chlorosis. Transcriptomic analysis suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

Abstract Background Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase. Results An engineered strain lacking a functional uptake hydrogenase (∆hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain. Conclusions Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

Abstract Background Most living cells uptake phosphate, an indispensable nutrient for growth from their natural environment. In Synechocystis sp. PCC 6803, the cells lack phosphate-inorganic transport (Pit) system but contain two phosphate-specific transport (Pst) systems, Pst1 and Pst2. We investigated the kinetics of Pi uptake of these two Pst systems by constructing the two mutants, ΔPst1 and ΔPst2, and comparing their kinetic properties with those of the wild-type cells under both Pi-sufficient and deficient conditions. The effects of pH and Na+ on the uptake of phosphate in Synechocystis were also studied. Results Growth rates of the two mutants and wild type were similar either under phosphate-sufficient or deficient condition. The Km for phosphate uptake was 6.09 μM in wild type and this was reduced to 0.13 μM in ΔPst1 cells and 5.16 μM in the ΔPst2 strain. The Vmax values of 2.48, 0.22, and 2.17 μmol • (min • mg of chlorophyll a)-1 were obtained for wild type, the ΔPst1 and ΔPst2 strains, respectively. A monophasic phosphate uptake was observed in wild-type cells. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects. Conclusion The data demonstrate that Pst2 system of Synechocystis has higher affinity toward phosphate with lower Vmax than Pst1 system. The Pst1 system had similar Km and Vmax values to those of the wild type suggesting that Pst1 is the main phosphate transporter in Synechocystis sp. PCC 6803. The Km of Pst1 of Synechocystis is closer to that of Pit system than to that of the Pst system of E. coli, suggesting that Synechocystis Pst1 is rather a medium/low affinity transporter whereas Pst2 is a high affinity transporter.

Various photoautotrophic cyanobacteria accumulate intracellular poly(3-hydroxybutyrate) (PHB) granules. This protocol can be used for determining the PHB contents of the cells as % PHB weight per dry cell weight using acid hydrolysis followed by high-performance liquid chromatography (HPLC). This HPLC analysis is rapid, with a running time of approximately 5 min per sample. The technique can accurately determine PHB concentrations in the range of 2–1,000 μg/mL PHB. However, this technique is not applicable for determining the contents of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in cyanobacteria.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99 µmol H2 g dry wt−1 and maximum hydrogenase activity of 179.39 ± 8.18 µmol H2 g dry wt−1 min−1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48 h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H₂ production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7 days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H₂ synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. Key message: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria. [ABSTRACT FROM AUTHOR]

The halotolerant cyanobacterium Aphanothece halophytica is a potential H2 producer that induces H2 evolution under nitrogen deprivation. H2 is mainly produced via the catabolism of stored glycogen under dark anaerobic condition. H2 evolution is catalyzed by O2-sensitive bidirectional hydrogenase. The aim of this study was to improve H2 production by A. halophytica using various kinds of inhibitors. Among all types of inhibitors, simazine efficiently promoted the highest H2 production under dark conditions. High simazine concentration and long-term incubation resulted in a decrease in cell and chlorophyll concentrations. The optimal simazine concentration for H2 production by A. halophytica was 25 µM. Simazine inhibited photosynthetic O2 evolution but promoted dark respiration, resulting in a decrease in O2 level. Hence, the bidirectional hydrogenase activity and H2 production was increased. A. halophytica showed the highest H2 production rate at 58.88 ± 0.22 µmol H2 g−1 dry weight h−1 and H2 accumulation at 356.21 ± 6.04 μmol H2 g−1 dry weight after treatment with 25 µM simazine under dark anaerobic condition for 2 and 24 h, respectively. This study demonstrates the potential of simazine for the enhancement of dark fermentative H2 production by A. halophytica.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni ®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 μmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.