Gold nanoparticles activate hydrogenase synthesis and improve heterotrophic growth of Ralstonia eutropha H16.

Ralstonia eutropha is a facultative chemolithoautotrophic aerobic bacterium that grows using organic substrates or H2 and CO2. Hydrogenases (Hyds) are synthesized under lithoautotrophic, or energy-limited heterotrophic conditions and are used in enzyme fuel cells (EFC) as anodic catalysts. The effects of chemically synthesized gold nanoparticles (Au-NPs) on R. eutropha H16 growth, oxidation-reduction potential (ORP) kinetics, and H2-oxidizing Hyd activity were investigated in this study. Atomic force microscopy showed that thin, plate-shaped Au-NPs were in the nanoscale range with an average size of 5.68 nm. Compared with growth in medium without Au-NPs (control), the presence of Au-NPs stimulated growth, and resulted in a decrease in ORP to negative values. H2-oxidizing activity was not detected in the absence of Au-NPs, but activity was significantly induced (12 U/g CDW) after 24 h of growth with 18 ng/ml, increasing a further 4-fold after 72 h of growth. The results demonstrate that Au-NPs primarily influence the membrane-bound Hyd. In contrast to R. eutropha, Au-NPs had a negligible or negative effect on the growth, Hyd activity, and H2 production of Escherichia coli. The findings of this study offer new perspectives for the production of oxygen-tolerant Hyds and the development of EFCs.

L-amino acids affect the hydrogenase activity and growth of Ralstonia eutropha H16.

Ralstonia eutropha H16 is a chemolithoautotrophic bacterium with O2-tolerant hydrogenase (Hyds) enzymes. Hyds are expressed in the presence of gas mixtures (H2, O2, CO2) or under energy limitation and stress conditions. O2-tolerant Hyds are promising candidates as anode biocatalysts in enzymatic fuel cells (EFCs). Supplementation of 0.5% (w/v) yeast extract to the fructose-nitrogen (FN) growth medium enhanced H2-oxidizing Hyd activity ~ sixfold. Our study aimed to identify key metabolites (L-amino acids (L-AAs) and vitamins) in yeast extract that are necessary for the increased synthesis and activity of Hyds. A decrease in pH and a reduction in ORP (from + 240 ± 5 mV to - 180 mV ± 10 mV values) after 24 h of growth in the presence of AAs were observed. Compared to the FN-medium control, supplementation of 7.0 µmol/ml of the L-AA mixture stimulated the growth of bacteria ~ 1.9 to 2.9 fold, after 72 h. The whole cells' H2-oxidizing Hyd activity was not observed in control samples, whereas the addition of L-AAs, mainly glycine resulted in a maximum of ~ 22 ± 0.5 and 15 ± 0.3 U, g CDW-1 activity after 24 h and 72 h, respectively. Our results suggest a correlation between ORP, pH, and function of Hyds in R. eutropha H16 in the presence of key L-AAs. L-AAs used in small amounts can be proposed as signaling molecules or key components of Hyd maturation. These results are important for the optimization of O2-tolerant Hyds production as anode biocatalysts.

Application of organic waste glycerol to produce crude extracts of bacterial cells and microbial hydrogenase-the anode enzymes of bio-electrochemical systems.

Glycerol is an organic waste material that can be used for the production of microbial biomass, consequently providing valuable biocatalysts promoting the generation of electrical current in microbial fuel cells (MFCs). [NiFe]-Hydrogenases (Hyds) of Escherichia coli and Ralstonia eutropha may be applied as potential anode biocatalysts in MFCs. In this study, E. coli K12 whole cells or crude extracts and R. eutropha HF649 synthesizing Strep-tagged membrane-bound Hyds (MBH) were evaluated as anode enzymes in a bioelectrochemical system. The samples were immobilized on the sensors with polyvinyl acetate support. Mediators like ferrocene and its derivatives (ferrocene-carboxy-aldehyde, ferrocene-carboxylic acid, methyl-ferrocene-methanol) were employed. The maximal level of bioelectrocatalytic activity of Hyds was demonstrated at 500 mV voltage. Depending on the mediator and biocatalyst, current strength varied from 5 to 42 µA. Introduction of ferrocene-carboxylic acid enhanced current strength; moreover, the current flow was directly correlated with H2 concentration. The maximal value (up to 150 µA) of current strength was achieved with a 2-fold hydrogen supply. It may be inferred that Hyds are efficiently produced by E. coli and R. eutropha grown on glycerol, while ferrocene derivatives act as agents mediating the electrochemical activity of Hyds.

Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity.

BACKGROUND: The chemolithoautotrophic ß-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. RESULTS: Growth, bioenergetic properties, and oxidation-reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L-1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)-1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N'-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. CONCLUSIONS: Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

Impact of membrane-associated hydrogenases on the F0F1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N'-dicyclohexylcarbodiimide in the mutants lacking hydrogenases.

Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH.

Redox stress in geobacilli from geothermal springs: Phenomenon and membrane-associated response mechanisms.

Geobacillus toebii ArzA-8, from Armenian geothermal springs, grew well in nutrient broth. During its growth, changes in pH in opposite directions were observed depending on glucose supplementation. Accordingly, the decrease in the redox potential was determined using titanium-silicate (Eh) and platinum (Eh') electrodes: Eh decreased to -150 ± 3 mV and Eh' to -350 ± 2 mV without glucose; the decrease in these potentials was smaller with glucose. Redox stress due to an oxidizer, K3[Fe(CN)6], or a reducer, dl-dithiothreitol (DTT), inhibited bacterial growth. However, a stimulatory effect of K3[Fe(CN)6] or DTT was observed with or without glucose, respectively. With glucose, the H(+) efflux was sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of FoF1FOF1-ATPase and other H(+) translocation mechanisms, but the addition of an oxidizer or reducer suppressed the H(+) efflux. The ATPase activity of membrane vesicles was ~1.3-fold higher in cells grown with glucose compared with cells grown without glucose. DCCD and DTT suppressed ATPase activity in cells grown without glucose, whereas DTT stimulated FOF1-ATPase activity in cells grown with glucose. Thus, G. toebii senses redox stress; this thermophile likely presents specific membrane-associated response mechanisms involving FOF1-ATPase to overcome redox stress and survive; these mechanisms are important for adaptation to extreme environments.

Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation.

Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical-the membrane potential (∆φ) and chemical-the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ∆φ (-130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pHex ∆φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H(+) transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H(+) efflux was determined to depend on pHex; overall and N,N'-dicyclohexylcarbodiimide (DCCD)-inhibitory H(+) efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pHex, and a role of Hyd enzymes in its generation is suggested.

Electromagnetic irradiation of Enterococcus hirae at low-intensity 51.8- and 53.0-GHz frequencies: changes in bacterial cell membrane properties and enhanced antibiotics effects.

Exposure to electromagnetic irradiation (EMI) of 51.8 and 53.0 GHz and low intensity (flux capacity of 0.06 mW cm(-2) ) for 1 h markedly decreased the energy-dependent H(+) and K(+) transport across membranes of Enterococcus hirae ATCC 9790. After EMI, there was also a significant decrease of overall and N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity of the membrane vesicles. These measures were considerably lower at 53.0 GHz. EMI in combination with different antibiotics, such as ceftriaxone and kanamycin at their minimal inhibitory concentrations (100 and 200 µM, respectively), enhanced bacterial cell growth and altered their membrane transport properties. Total H(+) efflux was most sensitive to ceftriaxone but DCCD-inhibited H(+) efflux and total K(+) influx were sensitive to kanamycin. The results indicate that cell membrane proteins could be a target in the action of EMI and enhanced antibacterial effects in combination with antibiotics. The DCCD-sensitive F(0) F(1) -ATPase or this ATPase in combination with K(+) uptake protein probably plays a key role in these effects.

Role of different Escherichia coli hydrogenases in H+ efflux and F1F(o)-ATPase activity during glycerol fermentation at different pH values.

Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild-type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.