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 (pH_ex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pH_ex at pH 7.5 and 6.5, respectively; and it was higher than pH_ex at pH 5.5. At high pH_ex, the increase of ?φ (?130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pH_ex ?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 pH_ex; 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 pH_ex, and a role of Hyd enzymes in its generation is suggested.     

Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H₂ production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H₂ production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H₂ production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)^?1 and Hyd-1 activity was 0.1 U (mg protein)^?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)^?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)^?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H₂ production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.     

Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h⁻¹. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-¹³C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-¹³C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (~95%) and redox (~96%) balances. On the other hand, cultivation conditions that prevent H₂ accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H₂ is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

End product inhibition in methane fermentations: Effects of carbon dioxide on fermentative and acetogenic bacteria

Summary The rates of glucose utilization by fermentative bacteria and propionate and butyrate utilization by acetogenic bacteria were studied and their dependence of pCO₂ in the interval 0–1 bar was determined. A batch fermentation method was used permitting good control of fermentation parameters and rapid experiments.The rate of glucose fermentation to acids, CO₂ and H₂ was in the order of 12,000 mg glucose/l · day which was about two orders of magnitude faster than the utilization of propionic and butyric acid by acetogenic bacteria. The rate of glucose utilization was about 30% greater at low values of pCO₂ compared with 1 bar CO₂.Propionate degradation was strongly affected by pCO₂; rates were 60 mg/l · day at pCO₂=1 bar and 200 mg/l · day at pCO₂=0.2 bar. Some CO₂ was required since the rate of propionate utilization dropped rapidly below pCO₂=0.2 bar. The rate of butyric acid utilization was constant at 170 mg/l · day; slightly lower at pCO₂=1 bar.Yields of methane from glucose or acids were close to the theoretical value 50% of degraded substrate-carbon. Yields were 20–30% higher at low values of pCO₂ compared with 1 bar CO₂.The redox potential was usually between –200 and –250 mV, slowly increasing to between –150 and –200 mV during fermentation. No clear connection between rates of substrate utilization, pCO₂ and E_h was detected.     

Cloning, expression, and characterization of coenzyme-B12-dependent diol dehydratase from Lactobacillus diolivorans

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α₂β₂γ₂) structure with a native molecular mass of 210 kDa. It requires coenzyme B₁₂ for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K _m values for coenzyme B₁₂, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B₁₂-dependent diol dehydratase and not a glycerol dehydratase.     

Direct potentiometric determination of redox potentials of the gut contents in the termites Zootermopsis nevadensis and Cubitermes severus and in three other arthropods

A Pt and calomel electrode combination were used to determine the redox potentials of the gut contents in two termites, Zootermopsis nevadensis and Cubitermes severus. Strongly reducing conditions occurred in the paunch of Z. nevadensis (mean E_h = ?160 mV), consistent with many evidences that anaerobic fermentation of wood polymers occurs at this site. In C. severus, a soil-feeder, equivalent regions of the hindgut were more midly reducing (P1: mean E_h = ?104 mV; P3: mean E_h = ?47 mV) while the colon appeared microaerobic or aerobic. It is argued that these conditions are more appropriate to the digestion of humic materials. Potentials consistent with aerobic conditions were found throughout the guts of Periplaneta americana, Locusta migratoria and Glomeris marginata, although the cockroach hindgut was more reducing than the equivalent structures in the other non-termite species.     

Metabolic fluxes in Schizosaccharomyces pombe grown on glucose and mixtures of glycerol and acetate

Growth on glycerol has already been a topic of research for several yeast species, and recent publications deal with the regulatory mechanisms of glycerol assimilation by the fission yeast Schizosaccharomyces pombe. We investigated glycerol metabolism of S. pombe from a physiological point of view, characterizing growth and metabolism on a mixture of glycerol and acetate and comparing it to growth on glucose under respirative growth conditions in chemostat experiments. On glycerol/acetate mixtures, the cells grew with a maximum specific growth rate of 0.11 h^?1 where 46 % of the carbon was channeled into biomass and the key fermentation product ethanol was not detectable. ¹³C-assisted metabolic flux analysis resolved substrate distributions through central carbon metabolism, proving that glycerol is used as a precursor for glycolysis, gluconeogenesis, and the pentose phosphate pathway, while acetate enters the tricarboxylic acid cycle via acetyl-CoA. Considering compartmentalization between cytosol and mitochondria in the metabolic model, we found compartmentalization of biosynthesis for the amino acids aspartate and leucine. Balancing of redox cofactors revealed an abundant production of cytosolic NADPH that must be finally regenerated via the respiratory chain shown by the simulated and measured CO₂ production and oxygen consumption rates which were in good agreement.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

H2 production from chemostat fermentation of glucose byClostridium butyricum andClostridium pasteurianum in ammonium- and phosphate limitation

Summary In ammonium-limitation (4.55 mM NH₄ ⁺) at a dilution rate (D)=0.081 h^–1,Clostridium butyricum produced 2 mol H₂ per mol glucose consumed at pH 5.0, but at a low fermentation rate. At higher pH, important amounts of extracellular protein were produced. Phosphatelimitation (0.5 mM PO₄ ^–3) at D=0.061 h^–1 and pH 7.0 were the best conditions tested for hydrogen gas production (2.22 mol H₂ per mol glucose consumed) at a high fermentation rate. Steady-state growth at lower pH and with 0.1 mM PO₄ ^–3 resulted in proportional higher glucose incorporation into biomass and lower H₂ production. C. pasteurianum in NH₄ ⁺ limitation showed higher fermentation rates thanC. butyricum and a stabilized H₂ production around 2.08 (±0.06) mol per mol glucose consumed at various defined pH conditions, although the acetate/butyrate ratio increased to 1 at pH 7.0. The latter was also observed in phosphate-limitation, but here H₂ production was maximal (1.90 mol. per mol glucose consumed) at the lowest pH (5.5) tested.     

The Effects of Manganese (II) But Not Nickel (II) Ions on Enterococcus hirae Cell Growth, Redox Potential Decrease, and Proton-Coupled Membrane Transport

Enterococcus hirae grow well under anaerobic conditions by fermenting glucose, accompanied with the decrease of oxidation–reduction potential (E _h) from positive values to negative ones. It was shown that heavy metals—copper and iron ions—affect E. hirae growth and alter E _h and proton-potassium ions fluxes through the cell membrane. The aim of this study was to establish the effects of manganese (II) ions on bacterial growth within the concentration range of 0.01–1 mM and compare with nickel (II) ions’ effect. The presence of Mn²⁺ during E. hirae ATCC9790 growth had significant effects: The lag phase duration decreased while the specific growth rate was increased; decrease in E _h was shifted. In contrast, no visible changes in bacterial growth and E _h were observed in the case of Ni²⁺. The effects of these ions on proton-potassium ions fluxes through the cell membrane were estimated in the presence and absence of N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. Stronger effect of Mn²⁺ on H⁺–K⁺ exchange was detected in the presence of DCCD that can be explained by a possible complex formation between these substances and its direct influence on membrane transport proteins.     

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO2/H2 blend

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.     

Effect of redox potential regulation on succinic acid production by Actinobacillus succinogenes

The effects of redox potential used as a control parameter on the process of succinic acid production in batch cultures of Actinobacillus succinogenes NJ113 have been investigated. In batch fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of ?100 to ?450 mV. From the results, the ORP level of ?350 mV was preferable, which resulted in high succinic acid yield (1.28 mol mol^?1), high succinic acid productivity (1.18 g L^?1 h^?1) and high mole ratio of succinic acid to acetic acid (2.02). The mechanism of redox potential regulation was discussed by metabolic flux analysis and the ratio of NADH/NAD⁺. We expected that redox potential can be used as a valuable parameter to monitor and control much more anaerobic fermentation production.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate,pH and the F0F1-ATPase

Molecular hydrogen (H₂) 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 H₂-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 F₀F₁-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H₂-oxidizing enzymes during glucose fermentation at neutral and low [pH]_out. They operate in a reverse, H₂-producing mode during glycerol fermentation at neutral [pH]_out. Hyd-1 and Hyd-2 activity depends on F₀F₁. Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H₂ production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H₂ production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H₂ production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81

Megasphaera elsdenii T81 grew on either dl-lactate or d-glucose at similar rates (0.85 h^?1) but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able to grow at much higher concentrations of d-glucose (500 mM), but never removed more than 80 mM of glucose from the medium, and nearly 60 % the glucose removed was sequestered as intracellular glycogen, with low yields of even-carbon acids (acetate, butyrate, caproate). In the presence of both substrates, glucose was not used until lactate was nearly exhausted, even by cells pregrown on glucose. Glucose-grown cultures maintained only low extracellular concentrations of acetate, and addition of exogenous acetate increased yields of butyrate, but not caproate. By contrast, exogenous acetate had little effect on lactate fermentation. At pH 6.6, growth rate was halved by exogenous addition of 60 mM propionate, 69 mM butyrate, 44 mM valerate, or 33 mM caproate; at pH 5.9, these values were reduced to 49, 49, 18, and 22 mM, respectively. The results are consistent with this species’ role as an effective ruminal lactate consumer and suggest that this organism may be useful for industrial production of volatile fatty acids from lactate if product tolerance could be improved. The poor fermentation of glucose and sensitivity to caproate suggests that this strain is not practical for industrial caproate production.     

Generation of electric potential difference across the electrodes of the microbial fuel cell in the anaerobic oxidation of substrates by microbial associations

Several microbial associations were obtained from natural and anthropogenic sources. All of the associations grow well on glucose and significantly worse on acetate. We observed 80–95% glucose consumption during 3–5 days of growth. The oxidation of substrates by the cultures generates an electric potential difference between anode and cathode electrodes of a microbial fuel cell (MFC). The value of the potential difference depends on the nature of the association and the substrate and reaches 400–500 mV. The potential difference generation accompanied by a shift to the negative region of the medium redox potential (E _h ) to — (400–500) mV. This indicates H₂ evolution by the association cultures during oxidation of carbohydrates. Artificial redox mediators, such as tetramethyl-p-phenylenediamine, phenazine methosulfate, and benzyl viologen, were able to increase up to 15% the difference in electrical potential across the electrodes of the MFC. It is assumed that an increase in the potential difference across the electrodes induced by the redox mediators is due to their direct involvement in the transfer of electrons from the bacteria in the incubation medium to the MFC anode electrode. The direct measurement of current and potential difference on the electrodes in a short-circuit mode shows that the internal resistance of the MFC is equal to 1 kΩ and the power reaches 5 μW. Undoubtedly, this testifies to the low efficiency developed by the MFE.     

Environmentally Safe Production of 7-ACA by Recombinant Acremonium chrysogenum

7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris–Cl (pH 9.6), supplement with 7 mM Zn²⁺, slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT–PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO₃ was reduced to 50 mM, ZnSO₄ was increased to 7 mM, CuSO₄ was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.