Hydrogen Production In Cyanobacteria Research Articles

Salt and heat stress enhances hydrogen production in cyanobacteria.

Cyanobacteria are among the most suitable organisms for the capture of excessive amounts of CO2 and can be grown in extreme environments. In our research we use the single-celled freshwater cyanobacteria Synechococcus elongatus PCC7942 PAMCOD strain and Synechocystis sp. PCC6714 for the production of carbohydrates and hydrogen. PAMCOD strain and Synechocystis sp. PCC6714 synthesize sucrose when exposed to salinity stress, as their main compatible osmolyte. We examined the cell proliferation rate and the sucrose accumulation in those two different strains of cyanobacteria under salt (0.4M NaCl) and heat stress (35 0C) conditions. The intracellular sucrose (mol sucrose content per Chl a) was found to increase by 50% and 108% in PAMCOD strain and Synechocystis sp. PCC6714 cells, respectively. As previously reported, PAMCOD strain has the ability to produce hydrogen through the process of dark anaerobic fermentation (Vayenos D, Romanos GE, Papageorgiou GC, Stamatakis K (2020) Photosynth Res 146, 235-245). In the present study, we demonstrate that Synechocystis sp. PCC6714 has also this ability. We further examined the optimal conditions during the dark fermentation of PAMCOD and Synechocystis sp. PCC6714 regarding H2 formation, increasing the PAMCOD H2 productivity from 2 nmol H2 h- 1 mol Chl a- 1 to 23 nmol H2 h- 1 mol Chl a- 1. Moreover, after the dark fermentation, the cells demonstrated proliferation in both double BG-11 and BG-11 medium enriched in NaNO3, thus showing the sustainability of the procedure.

Metabolic switching of central carbon metabolism in response to nitrate: Application to autofermentative hydrogen production in cyanobacteria

Nitrate removal from culture media is widely used to enhance autofermentative hydrogen production in cyanobacteria during dark anaerobiosis. Here we have performed a systematic inventory of carbon and nitrogen metabolites, redox pools, and excreted product fluxes which show that addition of nitrate to cultures of Synechococcus sp. PCC 7002 has no influence on glycogen catabolic rate, but shifts the distribution of excreted products from predominantly lactate and H2 to predominantly CO2 and nitrite, while increasing the total consumption of intracellular reducing equivalents (mainly glycogen) by 3-fold. Together with LC–MS derived metabolite pool sizes these data show that glycogen catabolism is redirected from the upper-glycolytic (EMP) pathway to the oxidative pentose phosphate (OPP) pathway upon nitrate addition. This metabolic switch in carbon catabolism is shown to temporally correlate with the pyridine nucleotide redox-poise (NAD(P)H/NAD(P)+) and demonstrates the reductant availability controls H2 evolution in cyanobacteria.

Fructose-Mediated Elevation of Hydrogen Production in Glucose Tolerant Mutant of Synechocystis Sp. Strain PCC 6803 under the Dark Anaerobic Nitrate-Free Condition

Fructose is a potential additive that elevates a supply of electrons to hydrogenase for hydrogen production in cyanobacteria. A series of dark anaerobic hydrogen production experiments was performed to evaluate the validity of this assumption, in which fructose from 0 to 110 mmol/L was added to HEPES buffer solution on which a glucose tolerant mutant of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (GT strain) was incubated. Despite the reported knowledge that fructose was an inhibitor for photoheterotrophic growth of Synechocystis cells, GT strain assimilated fructose and represented a limited heterotrophic growth on fructose in HEPES buffer solution under dark anaerobic condition. The initial hydrogen production rate that was 0.025 mmol/L h in run without fructose increased to 0.0917 mmol/L h in run with 60-83 mmol/L fructose. The associated increase in the initial amount of endogenous glucose from 0.22 mmol/L in run without fructose to 0.36 mmol/L in run with 50 mmol/L fructose was observed. Fructose released the complete suppression of hydrogen production by nitrate. This work presents the first experimental evidence that cells of GT strain are able to assimilate fructose for cell growth in dark anaerobic condition. Our results show that hydrogen production in Synechocystis sp. strain can be significantly elevated by a proper addition of fructose to dark anaerobic HEPES buffer solution.

Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH

Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.

Temperature-sensitive PSII and promiscuous PSI as a possible solution for sustainable photosynthetic hydrogen production

Sustainable hydrogen production in cyanobacteria becomes feasible as a result of our recent studies of the structure of photosystem I encoding operon in a marine phage. We demonstrated that the fused PsaJF subunit from the phage, substituted for the two separate subunits in Synechocystis, enabled the mutated PSI to accept electrons from additional electron donors such as respiratory cytochromes. In this way, a type of photorespiration was created in which the cell consumes organic material through respiratory processes and PSI serves as a terminal electron acceptor, substituting for cytochrome oxidase. We designed a hydrogen-producing bioreactor in which this type of photorespiration could utilize the organic material of the cell as an electron source for H2 production. We propose, in parallel, to engineer cyanobacterial and/or algal strains with a temperature-sensitive PSII and enhanced respiration rates to achieve efficient and sustainable hydrogen production. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N2. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the delta hupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the delta hupL delta nifV1 strain (with the hupL and nifV1 genes disrupted) and the delta hupL delta nifV2 strain, and a double mutant, the delta hupL delta nifV1 delta nifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent delta hupL strain. The hydrogen production activity of the delta hupL delta nifV1 mutant was sustained at higher levels than the activity of the parent delta hupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N2 gas inhibited hydrogen production in the delta hupL delta nifV1 delta nifV2 mutant less strongly than in the parent delta hupL strain and the delta hupL delta nifV1 and delta hupL delta nifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.

3-36.ラン色細菌のヒドロゲナーゼの分布とニトロゲナーゼを利用した光生物的水素生産((9)微生物等,Session 3 バイオマス等)

3-36.ラン色細菌のヒドロゲナーゼの分布とニトロゲナーゼを利用した光生物的水素生産((9)微生物等,Session 3 バイオマス等)

Surfactant‐Induced hydrogen production in cyanobacteria

Addition of Tween 85 to aqueous suspensions of Anabaena variabilis induced photosynthetic evolution of hydrogen over a time span of several weeks: As much as 148 nmol H(2)/h . mg dry weight was produced in the first week by a suspension containing 4.2 mg dry weight of cells and 77 mM Tween 85. The chemical structure of Tween 85 was a necessary prerequisite for inducing hydrogen production, as compounds such as Tween 20, 60, and 80 had a quite different effect. There was a coupling between photosynthetic oxygen evolution and hydrogen evolution: Hydrogen evolution started to be effective only when oxygen evolution subdued. The presence of heterocysts in A. variabilis was also required for the Tween-induced hydrogen production. Based on these observations, possible mechanisms for the photosynthetic effect of Tween 85 are advanced and discussed.