Interdependence of Escherichia coli formate dehydrogenase and hydrogen-producing hydrogenases during mixed carbon sources fermentation at different pHs

The impact of the four membrane-bound [NiFe]-hydrogenases (Hyd) of Escherichia coli on total H₂-oxidizing activity during fermentation of a mixture of glucose, glycerol and formate at different pHs was examined. It was shown that Hyd-2 had a major contribution to total Hyd activity at pH 7.5 in early-stationary phase (24 h) cells, while the main contribution was made by Hyd-3 in late-stationary phase (72 h). Hyd-4-dependent Hyd activity could be demonstrated at pH 6.5 in cells lacking Hyd-1, Hyd-2 and Hyd-3. at pH 7.5 Hyd-4-dependent formate dehydrogenase (FDH-H) activity was demonstrated. Growth properties and fermentation end product patterns during 72 h demonstrated that the cells retained viability deep into stationary phase. Our findings emphasize the importance of formate in modulating H₂ metabolism, presumably by contributing to maintain redox, pH and pmf balance. This is important for regulating and enhancing H₂ production when a mixture of carbon sources is applied.     

Enhancement of Escherichia coli bacterial biomass and hydrogen production by some heavy metal ions and their mixtures during glycerol vs glucose fermentation at a relatively wide range of pH

Escherichia coli growth and H₂ production were followed in the presence of heavy metal ions and their mixtures during glycerol or glucose fermentation at pH 5.5–7.5. Ni²⁺ (50 μM) with Fe²⁺ (50 μM) but not sole metals stimulated bacterial biomass during glycerol fermentation at pH 6.5. Ni²⁺+Fe³⁺ (50 μM), Ni² +Fe³⁺+Mo⁶⁺ (20 μM) and Fe³⁺+Mo⁶⁺ (20 μM) but not sole metals enhanced up to 3-fold H₂ yield but Cu⁺ or Cu²⁺ (100 μM) inhibited it. At pH 7.5 stimulating effect on biomass was observed by Ni²⁺+Fe²⁺+Mo⁶⁺. H₂ production was enhanced 2.7 fold particularly by Ni²⁺+Fe³⁺+Mo⁶⁺ at the late stationary growth phase. Whereas at pH 5.5 increased biomass was when Fe²⁺+Mo⁶⁺ or Mo⁶⁺ were added. H₂ yield was decreased compared with that at pH 6.5, but metal ions again enhanced it. During glucose fermentation at pH 6.5 biomass was increased by the mixtures of metal ions, and 1.2 fold increased H₂ yield was observed. At pH 7.5 Ni²⁺+Fe²⁺ increased biomass but Cu⁺ or Cu²⁺ had suppressing effect; Fe³⁺+Mo⁶⁺ stimulated H₂ production. At pH 5.5 biomass also was raised by Ni²⁺+Fe²⁺+Mo⁶⁺; H₂ yield was increased upon Mo⁶⁺ and Mo⁶⁺+Fe²⁺ or Mo⁶⁺+Fe³⁺ additions. The results point out the importance of Ni²⁺, Fe²⁺, Fe³⁺ and Mo⁶⁺ and some of their combinations for E. coli bacterial growth and H₂ production mostly during glycerol but not glucose fermentation and at acidic conditions (pH 5.5 and 6.5). They can be used for optimizing fermentation processes on glycerol, controlling bacterial biomass and developing H₂ production biotechnology.     

Hydrogen production by Escherichia coli growing in different nutrient media with glycerol: Effects of formate,pH, production kinetics and hydrogenases involved

The Escherichia coli BW25113 or MC4100 wild type parental strains growth and H₂ production kinetics was studied in batch cultures of minimal salt medium (MSM) and peptone medium (PM) at pH of 5.5–7.5 upon glycerol (10 g L^?1) fermentation and formate (0.68 g L^?1) supplementation. The role of formate alone or with glycerol on growth and H₂ production via hydrogenases (Hyd) was investigated in double hyaB hybC (lacking large subunits of Hyd 1 and 2), triple hyaB hybC hycE (lacking large subunits of Hyds 1-3) and sole selC (lacking formate dehydrogenase H) mutants during 24 h bacterial growth. H₂ production was delayed and observed after 24 h bacterial wild type strains growth on MSM. Moreover, it reached the maximal values after 72 h growth at the pH 6.5 and pH 7.5. Biomass formation of the mutants used was inhibited ～3.5 fold compared with wild type, and H₂ production was absent in hyaB hybC hycE and selC mutants upon glycerol utilization on MSM at pHs of 5.5–7.5. Formate inhibited bacterial growth on MSM with glycerol, but enhanced and recovered H₂ production by hybC mutant at pH 7.5. H₂ evolution was delayed at pH 7.5 in PM, but observed and stimulated at pH 6.5 upon glycerol and formate utilization in hyaB hybC mutant. H₂ production was absent in hyaB hybC hycE and selC mutants upon glycerol, formate alone or with glycerol fermentation at pH 6.5 and pH 7.5; formate supplementation had no effect. The results point out E. coli ability to grow and utilize glycerol in MSM with comparably high H₂ yield: as well as they suggest the key role of Hyd-3 at both pH 6.5 and pH 7.5 and the role of Hyd-2 and Hyd-4 at pH 7.5 in H₂ production by E. coli during glycerol fermentation with formate supplementation. The results obtained are novel and might be useful in H₂ production biotechnology development using different nutrient media and glycerol and formate as feedstock.     

Molecular Mechanisms of the Deregulation of Muscle Contraction Induced by the R90P Mutation in Tpm3.12 and the Weakening of This Effect by BDM and W7

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca²⁺-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca²⁺. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca²⁺-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca²⁺-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.     

Nonlinear rebound of a rod after impact against a deformable barrier

The nonlinear impact of a vehicle against a deformable barrier and its subsequent rebound from that barrier are simulated. A one-dimensional elastoplastic model represents the vehicle as a series of rod finite elements and the barrier as a single mass, nonlinear spring. The solution procedure utilizes variable time step integration, contains an error control and eliminates numerical instabilities. A limited study assesses the influence of system parameters on both structure and occupants. The chief objective, however, is to establish the feasibility of the proposed treatment of this class of problems. Some results obtained appear, at first, to contradict intuition. It is observed that modelling a deformable barrier as rigid can lead to unconservative results with respect to structural response of the vehicle.