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.     

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.     

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 wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities

In this study, the new strategy for long term bio-hydrogen (H₂) production using different substrates and waste materials is presented. Growth characteristics and H₂ production were investigated upon consumption of 0.4% xylose and 1% glycerol alone (which were optimal) or their mixture by Escherichia coli BW25113 wild type parental strain (PS) and ΔhyaB, ΔhybC, ΔhycE, ΔhyfG mutants with genes deletions for key subunits of hydrogenase (Hyd)-1 to Hyd-4, respectively, in high and low buffer capacity peptone (HPM, LPM) mediums, pH 5.5 and 7.5. Overall, pH 5.5 negatively affected bacterial growth and H₂ production. At pH 7.5, apart from Hyd-3 and Hyd-4 mutants, upon growth of PS, Hyd-1 and Hyd-2 mutants drop of Pt redox electrode readings from positive (～+150 mV) to negative (of ?400 to ?550 mV) values was detected during log growth phase mentioning H₂ formation. Xylose and glycerol co-utilization did not affect PS and Hyd-1 and Hyd-2 mutant's biomass and H₂ formation during log growth phase in LPM, but ～1.5 fold stimulated these parameters, especially in HPM, pH 7.5, during prolonged 96 h bacterial growth. Roles of Hyd-3 and Hyd-4 in H₂ production; and Hyd-1 and Hyd-2 in H₂ oxidation during bacterial log growth phase were stated under xylose and glycerol co-fermenting conditions. The results obtained might be valuable for industrial long term H₂ production by bacteria using mixture of carbon sources and combining various organic waste materials.     

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.     

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.     

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.     

Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs

Escherichia coli produces molecular hydrogen (H₂) during glucose or mixed carbon (glucose and glycerol) fermentation. Dependence of H₂ production rate (VH₂)$\left(V_{{\text{H}}_2}\right)$ on glucose at different pHs was studied in a concentration dependent manner. During growth of wild-type on glucose, increasing glucose concentration from 0.05% to 0.2% resulted in the marked inhibition of VH₂$V_{{\text{H}}_2}$. Inhibitory effect of glucose was shown at pH 7.5 and 6.5 but not pH 5.5. However, glycerol added in the growth medium with 0.1% glucose significantly increased VH₂$V_{{\text{H}}_2}$ but different effects at different pHs were established upon glucose or glycerol assays. The results indicate that H₂ production is inhibited by glucose in a concentration dependent manner during glucose fermentation but glucose in combination with glycerol might enhance H₂ production during mixed carbon fermentation.     

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.     

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.     

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.     

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.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

The effect of nutrient limitation on hydrogen production by batch cultures of Escherichia coli

In order to understand some limiting factors in microbial hydrogen fermentation we have examined hydrogen production by different strains of Escherichia coli grown in batch cultures under different limiting nutrient regimes. The effect of mutations in uptake hydrogenases, in lactate dehydrogenase (ldhA), and fhlA, coding for the regulator of formate hydrogen lyase (fhl) component synthesis, were studied. Each mutation contributed to a modest increase in hydrogen evolution and the effects were synergistic. Various elements were used as limiting nutrient. In batch experiments, limitation for sulfate was without great effect. There was some affect of limiting phosphate with yields approaching 1 mol per mol of glucose. However, strains showed the highest yield of hydrogen per glucose (∼2$\sim 2$) when cultured at limiting concentrations of either ammonia or glucose.     

Ni (II) and Mg (II) ions as factors enhancing biohydrogen production by Rhodobacter sphaeroides from mineral springs

Various metal ions play a key role in biohydrogen (H₂) production by phototrophic bacteria through incorporation into or stimulating the responsible enzymes and/or related pathways. The Ni (II) and Mg (II) ions effects on growth and H₂ production by Rhodobacter sphaeroides strain MDC6521 isolated from mineral springs in Armenia were established. The highest growth specific rate was obtained with 4–6 μM Ni²⁺ and 5 mM Mg²⁺. pH of the growth medium changed from 7.0 to 9.2–9.4 during the bacterial growth up to 72 h in spite of Ni²⁺ added but pH increased in different manner with Mg²⁺. In the presence of 2–4 μM Ni²⁺ external oxidation-reduction potential (ORP) decreased to more negative values (−800 ± 15 mV). This decrease of ORP indicated ∼2.7-fold enhanced H₂ yield (9.80 mmol L⁻¹) with Ni²⁺ compared with the control (without Ni²⁺). The H₂ yield determined in the medium with Mg²⁺ was ∼2.2 fold higher than that with 1 mM Mg²⁺. These results reveal new regulatory ways to improve H₂ production by R. sphaeroides those were depending on Ni²⁺ and Mg²⁺ of different concentrations.     

Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii cultures: The effect of the H2 partial pressure

Photoproduction of H₂ gas has been examined in sulfur/phosphorus-deprived Chalmydomonas reinhardtii cultures, placed in photobioreactors (PhBRs) with different gas phase to liquid phase ratios (V_g.p./V_l.p.). The results demonstrate that an increase in the ratio stimulates H₂ photoproduction activity in both algal suspension cultures and in algae entrapped in thin alginate films. In suspension cultures, a 4× increase (from ∼0.5 to ∼2) in V_g.p./V_l.p results in a 2× increase (from 10.8 to 23.1 mmol l⁻¹ or 264–565 ml l⁻¹) in the total yield of H₂ gas. Remarkably, 565 ml of H₂ gas per liter of the suspension culture is the highest yield ever reported for a wild-type strain in a time period of less than 190 h. In immobilized algae, where diffusion of H₂ from the medium to the PhBR gas phase is not affected by mixing, the maximum rate and yield of H₂ photoproduction occur in PhBRs with V_g.p./V_l.p above 7 or in a PhBR with smaller headspace, if the H₂ is effectively removed from the medium by continuous flushing of the headspace with argon. These experiments in combination with studies of the direct inhibitory effect of high H₂ concentrations in the PhBR headspace on H₂ photoproduction activity in algal cultures clearly show that H₂ photoproduction in algae depends significantly on the partial pressure of H₂ (not O₂ as previously thought) in the PhBR gas phase.     

Bioreactor for glycerol conversion into H2 by bacterium Enterobacter aerogenes

Glycerol was used as a substrate for H₂ production by bacterium Enterobacter aerogenes in the test tubes and bioreactor. A BioFlo/CelliGen 115 bioreactor (10 L working volume) was utilized to conduct the experiments for conversion of glycerol into H₂ by E. aerogenes cells. The highest H₂ production rate was observed under 2% glycerol in the culture medium. The glycerol uptake efficiency by bacteria in the bioreactor was found to be 65% during the 6 day period, matching glycerol uptake efficiency observed in the test tubes experiment (65%).Hydrogen production from glycerol (2% glycerol, v/v) by E. aerogenes in the bioreactor and test tubes was measured over the 6 days, showing the maximal H₂ rate at 650 mL g⁻¹ dry weight h⁻¹. The yield of H₂ production from glycerol at 0.89 mol/mol in the bioreactor was high, corresponding to the theoretical yield of 1 mol of H₂ per 1 mol of glycerol.