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.     

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.     

Sustained hydrogen production from formate using immobilized recombinant Escherichia coli SH5

Biohydrogen is an ideal energy carrier for mobile chemical fuel cells, but its use is often limited by unavailability of sustained H₂ production system(s). Here, we developed a compact system for H₂ production from formate based on immobilized cells of recombinant Escherichia coli SH5. Three different matrices were tested as immobilization medium, among which agar showed the best performance in mechanical stability and permeability of substrate(s) and/or gaseous products (H₂ and CO₂). To explore and optimize the H₂ production capability of the immobilized cells, the conditions for cell immobilization including cell loading and agar concentration as well as the factors affecting H₂ production rate such as temperature, pH, and substrate concentration were studied in detail. A maximum volumetric production rate of 2.4 L H₂ L⁻¹ h⁻¹ was obtained when the immobilized cells were incubated with 350 mM sodium formate at pH 6.5 and 37 °C. Periodic supplementation of 200 mM formate with 20 mM glucose at pH 6.5 maintained the high H₂ production rate for a prolonged period of 10 h. We believe that our process can be developed for sustained H₂ production and is applicable to the operation of fuel cells in small-scale.     

Prospecting hydrogen production of Escherichia coli by metabolic network modeling

Genome-scale model was applied to analyze the anaerobic metabolism of Escherichia coli. Three different methods were used to find deletions affecting fermentative hydrogen production: flux balance analysis (FBA), algorithm for blocking competing pathways (ABCP), and manual selection. Based on these methods, 81 E. coli mutants possessing one gene deletion were selected and cultivated in batch experiments. Experimental results of H₂ and biomass production were compared against the results of FBA. Several gene deletions enhancing H₂ production were found. Correctness of gene essentiality predictions of FBA for the selected genes was 78% and 77% in glucose and galactose media, respectively. 33% of the mutations that were predicted by FBA to increase H₂ production had a positive effect in experiments. Batch cultivation is a simple and straightforward experimental way to screen improvements in H₂ production. However, the ability of FBA to predict the H₂ production rate cannot be evaluated by batch experiments. Metabolic network models provide a method for gaining broader understanding of the complicated metabolic system of a cell and can aid in prospecting suitable gene deletions for enhancing H₂ 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.     

Fermentative hydrogen yields from different sugars by batch cultures of metabolically engineered Escherichia coli DJT135

Future sustainable production of biofuels will depend upon the ability to use complex substrates present in biomass if the use of simple sugars derived from food crops is to be avoided. Therefore, organisms capable of using a variety of fermentable carbon sources must be found or developed for processes that could produce hydrogen via fermentation. Here we have examined the ability of a metabolically engineered strain of Escherichia coli, DJT135, to produce hydrogen from glucose as well as various other carbon sources, including pentoses. The effects of pH, temperature and carbon source were investigated in batch experiments. Maximal hydrogen production from glucose was obtained at an initial pH of 6.5 and temperature of 35 °C. Kinetic growth studies showed that the μmax was 0.0495 h⁻¹ with a Ks of 0.0274 g L⁻¹ when glucose was the sole carbon source in M9 (1X) minimal medium. Among the many sugar and sugar derivatives tested, hydrogen yields were highest with fructose, sorbitol and d-glucose; 1.27, 1.46 and 1.51 mol H₂ mol⁻¹ substrate respectively.     

Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

H₂ production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H₂ production by fermentative bacteria by maximizing the number of electrons that are available for H₂. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O₂)-binding protein on oxic H₂ production in a closed batch system that was supplemented with glucose. The H₂ yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O₂ tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H₂ yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H₂ production and not by working as an O₂-scavenger during H₂ production. This new approach allowed more substrate to remain to be further utilized for the production of more H₂ from limited resources. We expect that VHb can be successfully engineered in potential aerobic H₂-producing microbial systems to enhance the overall H₂ production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H₂.     

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.     

Influence of growth curve phase on electricity performance of microbial fuel cell by Escherichia coli

The microbial fuel cell of Escherichia coli can convert microorganism biochemistry energy into electrical energy. To realize the influence of the growth curve phase with respect to different culture times on electricity performance, three kinds of E. coli (BCRC No. 10322, 10675, 51534) are selected, and it is both required and important to improve the performance of the microbial fuel cell (MFC). Results show that the BCRC No. 51534 of E. coli would be a better choice because a larger open-circuit voltage of 0.88 V and a limiting current of 10.1 mA possessed by it would result in an excellent power density of 547 mW/m². In addition, the selection of culture timing set as at the middle of the logarithmic phase and phase transition from logarithmic to stationary is suggested because the growth curve is suitable for electricity generation of the MFC. These observations would be useful for the improvement of the MFC.     

Hydrogen and ethanol fermentation of various carbon sources by immobilized Escherichia coli (XL1-Blue)

In this study, hydrogen and ethanol production by a facultative anaerobic bacterium Escherichia coli XL1-Blue immobilized in calcium-alginate beads have been investigated. Batch fermentations were carried out at mesophilic temperature (35 °C) and an initial cultivation pH of 6.5. Firstly, the influence of biomass concentration in terms of dry cell weight (expressed in g DCW/L, range 0.2–1.0) was investigated using fructose (5 g/L) as a carbon source. The peak hydrogen yield (HY) of 1.17 mol-H₂/mol-fructose_utilized was obtained at an initial cell concentration of 0.4 g DCW/L. The hydrogen production potential of other simple carbon sources (glucose and xylose) was evaluated at this optimized cell concentration and peak HY values were attained as 0.96 mol-H₂/mol-glucose_utilized and 0.69 mol-H₂/mol-xylose_utilized, respectively. In addition, utilization of the beverage wastewater (BWW) showed the peak cumulative hydrogen production and ethanol concentration of 120 mL and 5.65 g/L, attained at the substrate concentration of 20 g_{(glucose equivalent)}/L. However, peak HY (1.65 mol-H₂/mol-_{glucose eqivalent utilized}) was observed at low substrate concentration of 5 g_{(glucose equivalent)}/L. The percentage of sugar utilization of BWW was ranged between 80 and 96.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

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.     

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.     

Various hydrogenases and formate-dependent hydrogen production in Citrobacter amalonaticus Y19

An isolate Citrobacter amalonaticus   Y19 showed a typical mixed-acid fermentation with lactate and acetate as major end products when grown anaerobically on glucose and pyruvate, respectively. Production of hydrogen (_H2)$({\mathrm{H}}_2)$ from glucose, formate, and reduced methylviologen (MV) and benzylviologen (BV) by the resting cells of Y19 indicates the presence of formate hydrogen lyase (FHL) activity and other hydrogenases. Study with subcellular fractions of Y19 exhibited that the FHL activity, dependent on soluble formate dehydrogenase activity, was detected in the broken cell extract, but not in the soluble or particulate fraction which are separated by centrifugation at 35,000×g$35,000\times g$. Hydrogen production in the presence of reduced MV or BV was observed in both the soluble and particulate fractions. Uptake hydrogenase activity was observed in both the fractions when the oxidized forms of MV and BV were supplied as electron acceptor. In the soluble fraction, when formate was coupled with oxidized form of MV or BV, hydrogen production activity was recovered. These results indicate that, similar to E. coli, the strain Y19 expresses two different hydrogenases, one as the FHL complex and another as membrane-associated enzyme.     

Biohydrogen production from oil palm frond juice and sewage sludge by a metabolically engineered Escherichia coli strain

Biohydrogen is considered a promising and environmentally friendly energy source. Escherichia coli BW25113 hyaB hybC hycA fdoG frdc ldhA aceE has been previously engineered for elevated biohydrogen production from glucose. In this study, we show that this strain can also use biomass from oil palm frond (OPF) juice and sewage sludge as substrates. Substrate improvement was accomplished when hydrogen productivity increased 8-fold after enzymatic treatment of the sludge with a mixture of amylase and cellulase. The OPF juice with sewage sludge provided an optimum carbon/nitrogen ratio since the yield of biohydrogen increased to 1.5 from 1.3 mol H₂/mol glucose compared to our previous study. In this study, we also reveal that our engineered strain improved 200-fold biohydrogen productivity from biomass sources compared to the unmodified host. In conclusion, we determined that our engineered strain can use biomass as an alternative substrate for enhanced biohydrogen production.     

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H₂). CO-dependent H₂ production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H₂ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein⁻¹) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein⁻¹). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein⁻¹. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H₂ from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H₂ase for CO-dependent H₂ production.     

Hydrogen production by Escherichia coli without nitrogen sparging and subsequent use of the waste culture for fast mass scale one-pot green synthesis of silver nanoparticles

In view of the transition to hydrogen as a major energy carrier in the future, new routes for bringing down the cost of biological hydrogen production need to be explored. The current study was devoted to optimizing the dark fermentation by Escherichia coli HD701 for hydrogen production from an acid-hydrolyzed potato starch residue stream without nitrogen sparging to reduce the cost. To further increase the economic feasibility of hydrogen production by E. coli, this study explores the use of the waste culture after hydrogen production in mass scale one-pot green synthesis of silver nanoparticles.