Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

Lactobacilli isolated from various sources were identified on the basis of 16S -23S rRNA gene intergenic region amplification and subsequent sequencing of the smaller intergenic region. An in vitro analysis of probiotic properties including binding, ability to tolerate different concentrations of bile, survival in acidic buffer and antimicrobial activity of four different isolates and two standard strains (Lactobacillus plantarum American Type Culture Collection (ATCC) 8014 and L. rhamnosus GG (LGG)) was carried out. The ability of each isolate to stimulate Caco-2 cells, human peripheral blood mononuclear cells (PBMC) and THP-1 cells resulting in immunomodulation of these cells was analysed. Isolates L. rhamnosus CS25 and L. delbrueckii M and standard strain ATCC 8014 showed broad antimicrobial activity, and isolates CS25 (percentage of survival 6·9 % at pH 2·5, 5·1 % at pH 2·0) and L. plantarum CS23 (5·7 % at pH 2·5, 4·9 % at pH 2·0) have shown good tolerance to acidic pH. Isolate CS23 showed a good survival (14 %) after 2 h incubation in de Man, Rogosa and Sharpe (MRS) medium containing 3 % bile salts. Isolates CS23, CS25 and L. fermentum ASt1 could stimulate Caco-2 cells, human PBMC and THP-1 cells for a strong and varied immunomodulatory response in these cells. Though LGG showed poor antimicrobial activity as well as bile and acid tolerance, it was found to be the best binding strain tested. Child faecal isolate CS23 from the present study showed high binding ability (seventeen bacteria/Caco-2), high tolerance to acidic pH and bile salts and significant immunomodulation; therefore it is a good potential probiotic candidate.

No abstract

Nitrogen fixing cyanobacteria are being increasingly explored for nitrogenase-dependent hydrogen production. Commercial success however will depend on the ability to grow these cultures at high cell densities. Photo-limitation at high cell densities leads to hindered photoautotrophic growth while turbulent conditions, which simulate flashing light effect, can lead to oxygen toxicity to the nitrogenase enzyme. Cyanothece sp. strain ATCC 51142, a known hydrogen producer, is reported to grow and fix nitrogen under moderately oxic conditions in shake flasks. In this study, we explore the growth and nitrogen fixing potential of this organism under turbulent conditions with volumetric oxygen mass transfer coefficient (KL a) values that are up to 20-times greater than in shake flasks. In a stirred vessel, the organism grows well in turbulent regime possibly due to a simulated flashing light effect with optimal growth at Reynolds number of approximately 35,000. A respiratory burst lasting for about 4 h creates anoxic conditions intracellularly with near saturating levels of dissolved oxygen in the extracellular medium. This is concomitant with complete exhaustion of intracellular glycogen storage and upregulation of nifH and nifX, the genes encoding proteins of the nitrogenase complex. Further, the rhythmic oscillations in exhaust gas CO2 and O2 profiles synchronize faithfully with those in biochemical parameters and gene expression thereby serving as an effective online monitoring tool. These results will have important implications in potential commercial success of nitrogenase-dependent hydrogen production by cyanobacteria.

Cyanobacteria, a group of photosynthetic prokaryotes, oscillate between day and night time metabolisms with concomitant oscillations in gene expression in response to light/dark cycles (LD). The oscillations in gene expression have been shown to sustain in constant light (LL) with a free running period of 24 h in a model cyanobacterium Synechococcus elongatus PCC 7942. However, equivalent oscillations in metabolism are not reported under LL in this non-nitrogen fixing cyanobacterium. Here we focus on Cyanothece sp. ATCC 51142, a unicellular, nitrogen-fixing cyanobacterium known to temporally separate the processes of oxygenic photosynthesis and oxygen-sensitive nitrogen fixation. In a recent report, metabolism of Cyanothece 51142 has been shown to oscillate between photosynthetic and respiratory phases under LL with free running periods that are temperature dependent but significantly shorter than the circadian period. Further, the oscillations shift to circadian pattern at moderate cell densities that are concomitant with slower growth rates. Here we take this understanding forward and demonstrate that the ultradian rhythm under LL sustains at much higher cell densities when grown under turbulent regimes that simulate flashing light effect. Our results suggest that the ultradian rhythm in metabolism may be needed to support higher carbon and nitrogen requirements of rapidly growing cells under LL. With a comprehensive Real time PCR based gene expression analysis we account for key regulatory interactions and demonstrate the interplay between clock genes and the genes of key metabolic pathways. Further, we observe that several genes that peak at dusk in Synechococcus peak at dawn in Cyanothece and vice versa. The circadian rhythm of this organism appears to be more robust with peaking of genes in anticipation of the ensuing photosynthetic and respiratory metabolic phases.