Proton conductance and regulation of proton/potassium fluxes in Escherichia coli FhlA-lacking cells during fermentation of mixed carbon sources.

Escherichia coli uptake potassium ions with the coupling of proton efflux and energy utilization via proton FOF1-ATPase. In this study contribution of formate hydrogen lyase (FHL) complexes in the proton/potassium fluxes and the formation of proton conductance (CMH+) were investigated using fhlA mutant strain. The proton flux rate (JH+) decreased in fhlA by âˆ¼ 25 % and ∼70 % during the utilization of glucose and glycerol, respectively, at 20 h suggesting H+ transport via or through FHL complexes. The decrease in JK+ in fhlA by ∼40 % proposed the interaction between FHL and Trk secondary transport system during mixed carbon fermentation. Moreover, the usage of N,N'-dicyclohexylcarbodiimide (DCCD) demonstrated the mediation of FOF1-ATPase in this interaction. CMH+ was 13.4 nmol min-1 mV-1 in WT at 20 h, which decreased by 20 % in fhlA. Taken together, FHL complexes have a significant contribution to the modulation of H+/K+ fluxes and the CMH + for efficient energy transduction and regulation of the proton motive force during mixed carbon sources fermentation.

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.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37 °C, pH 7.5 and 30 °C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5 mmol/L and cumulative H2 ~ 94 mL g/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable ß-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable ß-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

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.

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.

Hydrogen-oxidizing hydrogenases 1 and 2 of Escherichia coli regulate the onset of hydrogen evolution and ATPase activity, respectively, during glucose fermentation at alkaline pH.

Simultaneous measurement of redox potential (Eh ) and determination of H2 evolution kinetics using a pair of titanium silicate and platinum redox electrodes in fermenting cultures of Escherichia coli wild type and different mutants lacking hydrogenases 1 (Hyd-1) or 2 (Hyd-2) revealed that Hyd-1 controls the onset of H2 evolution at slightly alkaline pH (pH 7.5) and under oxidizing Eh . In addition, Hyd-2 influences the N,N'-dicyclohexylcarbodiimide-inhibited ATPase activity in fermenting cells and thus regulates the proton F0 F1 -ATPase at the alkaline pH but under reducing Eh .

Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pHs.

Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E ( h )). During glycerol utilization at pH 7.5 ∆pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E ( h ) dropped down to negative values (-150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H(2) was evolved at the middle log phase while during glucose fermentation H(2) was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H(2) formation. Reducing reagents DL-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H(2) production. The findings point out the importance of reductive conditions for glycerol fermentation and H(2) production by E. coli.

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase.

Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.

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.

The increase in the number of accessible SH-groups in the enterococcal membrane vesicles by ATP and nicotinamide adenine dinucleotides.

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD(+) +NADH) to the vesicles caused a approximately 4-fold or approximately 1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD(+) +NADH both were added. The change was lacking in the presence of the F0F1-ATPase inhibitors N,N'-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F0F1-ATPase and, in addition, it was less in potassium ion-free medium. These results are correlated with data about K+-dependent F0F1-ATPase activity, suggesting a relationship between the F0F1-ATPase and K+ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.