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.     

Evidence for hydrogenase-4 catalyzed biohydrogen production in Escherichia coli

Biohydrogen production by Escherichia coli during fermentation of the mixture of glycerol, glucose and formate at different pH values was studied. Employing mutants lacking large subunits of different hydrogenases (Hyd), it was reported that, at pH 7.5, H₂ production was produced except in a hyaB hybC hycE triple mutant, thus suggesting compensatory H₂-producing functions of the Hyd enzymes. Activity of Hyd-4 was revealed in glucose assays at pH 7.5 in the triple mutant whereby 62% of the wild type level of H₂ production was derived from Hyd-4. In formate assays, it was shown, that, first, the hyaB hybC double mutant had a H₂ production ～3 fold higher than wild type, indicating that Hyd-1 and Hyd-2 oxidize H₂, and second, that at pH 5.5, Hyd-4 and Hyd-3 were responsible for H₂ production. These findings are significant when applying various carbon sources such as sugars, alcohol and organic acids for biohydrogen production.     

The influence of hydrogen production on the formation of metabolic pathways and regulation of ΔpH in Escherichia coli

Hydrogen (H₂) metabolism in Escherichia coli occurs via reversible membrane-associated hydrogenase enzymes (Hyd). Hyd-3 and Hyd-4 with formate dehydrogenase H (FDH-H) form formate hydrogen lyase complexes. The changes of metabolic pathways and ΔpH (pH_in-pH_ex) regulation during fermentation of glucose, glycerol and formate in non H₂-producing hypF (lack of all Hyds) and fdhF (lack of FDH-H) mutants at pH 7.5 were investigated. It was shown that specific growth rate was higher by ～23% in hypF and fdhF, compared to wild type (wt), suggesting the negative effect of H₂ on bacterial growth. Moreover, it was shown that H₂ generation did not have a vital role in glucose and glycerol utilization rate at 0–72 h. The utilization of external formate was detected in wt (～2.6 mM) and hypF (～0.68 mM), but not in fdhF, due to the absence of enzyme responsible for formate metabolism. Nevertheless, the changes in ΔpH were not evident at 3 h. The ratio of generated end-products and regulation of ΔpH at late log (6 h) and exponential phase (24, 72 h) were various in hypF and fdhF due to formate disproportionation in hypF and proton generation, therewith absence of H₂ generation. Taken together it can be concluded that bacteria regulate generation of fermentation end-products via balancing the concentration of acids and ethanol to maintain ΔpH and redox potential values. The results obtained are important for development and regulation of H₂ production technology when applying mixed carbon sources.     

Biomass and biohydrogen production during dark fermentation of Escherichia coli using office paper waste and cardboard

Growth properties, oxidation-reduction potential kinetics and hydrogen production of Escherichia coli BW25113 parental strain (PS) and hydrogenase (Hyd)-negative mutants were investigated after fermentative growth using office paper waste and cardboard (PW) hydrolysate (PWH). PWH was obtained by using dilute acid method in a steam sterilizer for 1 h, 121 °C. Optimal conditions for bacterial growth and H₂ production were identified (PWH concentrations, pH 7.5). Recording of redox potential using a platinum electrode revealed a drop to −500 ± 10 mV, with a H₂ yield of ~1.45 mmol H₂ L⁻¹ after 4 h of growth using PWH resulted in the formation of 0.20 ± 0.02 g bacterial cell dry weight L⁻¹. Bacterial biomass formation was stimulated ~3-fold upon addition of 0.5% yeast extract, and H₂ production started early - at the beginning of the exponential phase. Moreover, mutants lacking Hyd-1 and Hyd-2 significantly enhanced H₂ production. The findings would be beneficial for the development of H₂ production biotechnology using cheap solid waste materials.     

Escherichia coli hydrogenase 4 (hyf) and hydrogenase 2 (hyb) contribution in H2 production during mixed carbon (glucose and glycerol) fermentation at pH 7.5 and pH 5.5

Molecular hydrogen (H₂) production by Escherichia coli was studied during mixed carbon sources (glucose and glycerol) fermentation at pH 7.5 and pH 5.5. H₂ production rate (V_H2) by bacterial cells grown on mixed carbon was assayed with either adding glucose (glucose assay) or glycerol (glycerol assay) and compared with the cells grown on sole carbon (glucose or glycerol only) and appropriately assayed. Wild type cells grown on mixed carbon, in the assays with adding glucose, produced H₂ at pH 7.5 with the same level as in the cells grown on glucose only. At pH 7.5 V_H2 in fhlA single and fhlA hyfG double mutants decreased ∼6.5 and ∼7.9 fold, respectively. In wild type cells grown on mixed carbon V_H2 at pH 5.5 was lowered ∼2 fold, compared to the cells grown on glucose only. But in hyfG and hybC single mutants V_H2 was decreased ∼2 and ∼1.6 fold, respectively. However, at pH 7.5, in the assays with glycerol, V_H2 was low, when compared to the cells grown on glycerol only. At pH 5.5 in the assays with glycerol V_H2 was absent. Moreover, V_H2 in wild type cells was inhibited by 0.3 mM N,N^′-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F₀F₁-ATPase, in a pH dependent manner. At pH 7.5 in wild type cells V_H2 was decreased ∼3 fold but at pH 5.5 the inhibition was ∼1.7 fold. At both pHs in fhlA mutant V_H2 was totally inhibited by DCCD. Taken together, the results obtained indicate that at pH 7.5, in the presence of glucose, glycerol can also be fermented. They point out that Hyd-4 mainly and Hyd-2 to some extent contribute in H₂ production by E. coli during mixed carbon fermentation at pH 5.5 whereas Hyd-1 is only responsible for H₂ oxidation.     

Defining the roles of the hydrogenase 3 and 4 subunits in hydrogen production during glucose fermentation: A new model of a H2-producing hydrogenase complex

Hydrogen (H₂)-producing hydrogenase (Hyd) activity of E. coli wild type and mutants with defects in subunits of Hyd-3 or Hyd-4 during fermentation at different glucose concentrations and pHs was studied. Hyd-3 was mainly responsible for H₂ production but a significant contribution by Hyd-4 to total H₂ production depended on the glucose concentration and pH. Surprisingly, not all Hyd-3 or Hyd-4 subunits contributed towards H₂ production. Hyd-4 mainly exhibited H₂-oxidizing activity in cells growing on 0.2% glucose at pH 7.5, while at pH 5.5 it had a significant impact on H₂ production. Importantly, a hyfG mutant (lacking the large subunit of Hyd-4) had a ~2.2 fold decrease in H₂ production when cells were grown with 0.2% glucose. A similar role of Hyd-4 was shown at pH 6.5 grown with 0.8% glucose. This study provides new information to allow improvements in H₂ production yield and in our general understanding of H₂ metabolism.     

Escherichia coli hydrogenase activity and H2 production under glycerol fermentation at a low pH

Hydrogenase (Hyd) activity and H₂ production by Escherichia coli were studied at a low pH. H₂ production at pH 5.5 under glycerol fermentation was shown to be ∼1.5-fold higher than that at pH 6.5 or above but less than that under glucose fermentation. It was inhibited by N,N′-dicyclohexylcarbodiimide: H₂ production inhibition was increased with decreasing pH and almost maximal inhibition was observed at pH 5.5. The data on H₂ production by single and double mutants with defects in different Hyd-enzymes and in fhlA gene suggest that under glycerol fermentation at a low pH, Hyd-1, Hyd-2 and Hyd-4 were operating in a reversed, non-H₂ producing mode. Moreover, a role of fhlA gene in Hyd-3 and Hyd-4 activity in H₂ production is proposed under glucose fermentation at a low pH.     

Prolongation of H2 production during mixed carbon sources fermentation in E. coli batch cultures: New findings and role of different hydrogenases

Hydrogen (H₂) gas production in batch cultures was studied upon utilization of the mixture of glucose, glycerol and formic acid by Escherichia coli BW25113 wild type (wt) at pH of 5.5–7.5. At pH 7.5H₂ was continuously produced during 240 h but at pH 6.5 and 5.5 it was detected till 168 h and 120 h, respectively. Specific growth rate (μ) of wt was the highest (1.05 h^?1) at pH 6.5. Moreover, at pH 5.5 in hycE μ decreased by ～4.14 fold compared to wt, suggesting major role of Hyd-3 in cell growth. H₂ yield (8.8 mmol H₂ L^?1) was the highest at pH 7.5. In hybC H₂ yield was increased ～1.62 fold than in wt. These data might be applied for biomass and biohydrogen production from various organic wastes where mixtures of carbon sources are present.     

Enhanced hydrogen gas production from mixture of beer spent grains (BSG) and distiller's grains (DG) with glycerol by Escherichia coli

Escherichia coli perform mixed acid fermentation and produce hydrogen gas (H₂) as one of the fermentation end products. E. coli can ferment sugars like glucose, xylose and alcohols like glycerol. It has been shown that E. coli has the ability to utilize pretreated organic waste (BSG or DG) or mixtures of it with glycerol and H₂ can be produced. H₂ evolution was maximum when the concentration of BSG was 4% and DG - 10% yielding 1.4 mmol L⁻¹ H₂. H₂ evolution was prolonged to ~24–120 h when mixtures of glycerol and DG or BSG wastes were applied. Moreover, in hycE (lacking large subunit of Hyd-3) or hyfG (lacking large subunit of Hyd-4) single mutants H₂ production was absent compared to wild type suggesting that Hyd-3 and Hyd-4 are responsible for H₂ generation. In addition, multiple mutant enhanced cumulative H₂ production ~3–4 fold. Taken together it can be proposed that BSG or DG wastes either together or in mixture with glycerol can be applied to obtain E. coli biomass and produce bio-H₂. The novel data can be used to further control effectively the application of organic waste resources as a feedstock for developing bio-H₂ production technology.     

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.     

Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield,prolonged production

Escherichia coli wild type has the ability to utilize lactose or the mixture of lactose and glycerol producing bio-hydrogen (H₂) at different pH values. At pH 7.5 in hyaB (lacking large subunit of hydrogenase (Hyd)-1) and hybC (lacking large subunit of Hyd-2) single mutants fermenting lactose (1 g L⁻¹) H₂ yield was ∼7- and 5-fold more, respectively, compared to the wild type. During the fermentation of lactose (1 g L⁻¹) and glycerol (10 g L⁻¹) mixture H₂ yield in wild type increased ∼3-fold, compared to fermenting lactose only. H₂ generation in wild type was monitored in batch cultures during 168 h of growth when utilizing the mixture of lactose and glycerol in all combinations of different concentration. In hyaB but not in hybC mutant H₂ evolution was detected till 240 h in the mixture of 5 g L⁻¹ lactose and 10 g L⁻¹ glycerol. The highest H₂ production rate of 21.94 mL L⁻¹ h⁻¹ was detected in hyaB mutant at pH 7.5 when 1 g L⁻¹ lactose was applied. The results showed optimized H₂ production using different mutants, lactose and its mixture with glycerol. They can be applied for renewable energy, especially bio-H₂ production.     

Evaluation of hydrogen metabolism by Escherichia coli strains possessing only a single hydrogenase in the genome

The four hydrogenase isozymes; hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) of Escherichia coli have been reported for their crucial functions in the hydrogen metabolism; however, their distinctive roles could not be completely understood. In this study, four ideal hydrogenase operon mutants, Δhyb hyc hyf, Δhya hyc hyf, Δhya hyb hyf, and Δhya hyb hyc, in which only a single hydrogenase is intact in the genome, were constructed as well as one quadruple mutant (Δhya hyb hyc hyf) that all four hydrogenase operons were deleted. First, single operon mutants and single-gene mutants for each hydrogenase showed different hydrogen productivity and growth in the anaerobic fermentation, indicating that bacterial phenotype regarding the hydrogen metabolism via the deletion of each operon is different with that of each single gene. Then, 4 triple hydrogenase operon mutants and one quadruple mutant were investigated to evaluate the hydrogen metabolism (hydrogen production and uptake) using glucose or glycerol as a substrate of hydrogen fermentation. With both the carbon sources, only Hyd-2 and Hyd-3 were able to produce hydrogen. Furthermore, all the hydrogenases showed hydrogen uptake activity. In addition, no hydrogen production and hydrogen uptake were detected in the quadruple mutant which does not have all 4 hydrogenases. Hydrogen production from Hyd-2 and Hyd-3 was further confirmed by complementing their operons in the cloning vector pBR322.     

Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of various heavy metals on hydrogenase activity

Hydrogen is a carbon-neutral energy feedstock which is produced during fermentation of various carbon sources. The genomes of clostridia encode mainly [Fe-Fe]-hydrogenases. Clostridium beijerinckii DSM791 performed anaerobic fermentation of glycerol in batch culture at pH 7.5 and pH 5.5 and produced H₂. At pH 7.5, the glycerol consumption rate was 3.7 g/g cell mass/h, which was higher than that at pH 5.5. H₂ production reached 5 mmol/h/g cell mass at pH 7.5. The specific hydrogenase activity was ～1.4 fold higher if cells were grown on glycerol compared to cells grown on glucose. Single (Fe²⁺, Fe³⁺, Ni²⁺) or mixed supply of metals (Fe²⁺ and Ni²⁺) increased the specific hydrogenase activity by ～50%. These results suggest that C. beijerinckii DSM791 could be used as a potential H₂ producer. It may help to further enhance H₂ production using different industrial or agricultural wastes where glycerol and other carbon sources are present.     

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.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Different role of focA and focB encoding formate channels for hydrogen production by Escherichia coli during glucose or glycerol fermentation

The dependence of H₂ production on the formate channels, FocA and FocB, by Escherichia coli at pH 5.5, 6.5 and 7.5 was shown using focA and focB mutants and comparing with the wild type. Moreover, effect of exogenous addition of formate (10 mM) on H₂ production was allotted. The results acquired propose that during glucose fermentation formate import can occur through FocB at different pHs; external formate drives FocA to import direction. However, during glycerol fermentation formate might be imported through FocB, whereas formate is exported preferentially through FocA at pH 7.5.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration

Escherichia coli produces molecular hydrogen (H₂) during glucose fermentation. This production of H₂ occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H₂ producing rate (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ by Hyd-4 (hyf) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621 (hyfB-R  ) mutants (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was decreased ∼6.7 and ∼5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was severely decreased ∼7.8 and ∼3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H₂ producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H₂ production by E. coli during fermentation.     

Inoculum pretreatment differentially affects the active microbial community performing mesophilic and thermophilic dark fermentation of xylose

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H₂ production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H₂ mol^?1 xylose. The H₂ and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H₂ production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H₂ mol^?1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum.