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.     

Genome based metabolic flux analysis of Ethanoligenens harbinense for enhanced hydrogen production

Ethanoligenens harbinense is a promising hydrogen producing microorganism due to its high inherent hydrogen production rate. Even though the effect of media optimization and inhibitory metabolites has been studied in order to improve the hydrogen productivity of these cultures, the identification of the underlying causes of the observed changes in productivity has not been targeted to date. In this work we present a genome based metabolic flux analysis (MFA) framework, for the comprehensive study of E. harbinense in culture, and the effect of inhibitory metabolites and media composition on its metabolic state. A metabolic model was constructed for E. harbinense based on its annotated genome sequence and proteomic evidence. This model was employed to perform MFA and obtain the intracellular flux distribution under different culture conditions. These results allow us to identify key elements in the metabolism that can be associated to the observed production phenotypes, and that can be potential targets for metabolic engineering in order to enhanced hydrogen production in E. harbinense.     

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.     

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.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.     

How bacteria get energy from hydrogen: a genetic analysis of periplasmic hydrogen oxidation in Escherichia coli

Dihydrogen oxidation is an important feature of bacterial energy conservation. In Escherichia coli hydrogen oxidation (‘uptake’) is catalysed by membrane-bound [NiFe] hydrogenase-1 and [NiFe] hydrogenase-2. The bulk of these uptake isoenzymes is exposed to the periplasm and biosynthesis of the proteins involves membrane transport via the twin-arginine translocation (Tat) pathway. Hydrogenase-2 is encoded by the hybOABCDEFG operon and the core enzyme is a heterodimer of HybO and HybC. HybO is synthesised with a twin-arginine signal peptide. HybOC is associated with two other proteins (HybA and HybB) that complete the respiratory complex. The HybOC dimer is bound to the cytoplasmic membrane and appears to be anchored via a hydrophobic transmembrane α-helix located at the C-terminus of HybO. Thus, hydrogenase-2 is an example of an integral membrane protein assembled in a Tat-dependent (Sec-independent) manner. Studies of the biosynthesis, targeting, and assembly of hydrogenase-2 would set a paradigm for all respiratory complexes of this type.     

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.     

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.     

Field solar E. coli inactivation in the absence and presence of TiO2: is UV solar dose an appropriate parameter for standardization of water solar disinfection?

Evaluation of solar treatment in the absence and presence of TiO₂ has been made to assess its effectiveness in reducing bacterial load with respect to drinking water standards.Field experiments under direct solar radiation were carried out using a compound parabolic collector (CPC) placed at the Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland. Water contaminated with E. coli K12 was exposed to sunlight in different seasons. The obtained results indicate that the presence of TiO₂ accelerates the detrimental action of light. Total photocatalytic disinfection was obtained in both periods of year and no bacterial recovery was observed during 24 h after stopping sunlight exposure. In the absence of TiO₂, total disinfection was not always reached; and bacterial recovery was observed, especially when inactivation was not complete. Bacterial decay was mainly dependent on light intensity. It was also demonstrated that solar UV dose is not a pertinent parameter to standardize solar disinfection. The influence of the following topics on solar water disinfection is also studied in this paper: (a) UV and total solar spectra characteristics (b) volume of phototreated water (c) post-irradiation events.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

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.     

Engineering strategies for the enhanced photo-H2 production using effluents of dark fermentation processes as substrate

The major obstacle of combining dark and photo fermentation for high-yield biohydrogen production is substrate inhibition while using dark fermentation effluent as the sole substrate. To solve this problem, the dark fermentation broth was diluted with different dilution ratio to improve photo-H₂ production performance of an indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5. The best photo-H₂ production performance occurred at a dilution ratio of 1:2, giving a highest overall H₂ production rate of 10.72 ml/l/h and a higher overall H₂ yield of 6.14 mol H₂/mol sucrose. The maximum H₂ content was about 88.1% during the dilution ratio of 1:2. The photo-H₂ production performance was further improved by supplying yeast extract and glutamic acid as the nutrient. The results indicate that the overall H₂ production rate and H₂ yield increased to 17.02 ml/l/h and 10.25 mol H₂/mol sucrose, respectively. Using a novel solar-energy-excited optical fiber photobioreactor (SEEOFP) with supplementing tungsten filament lamp (TL) irradiation, the overall H₂ production rate was improved to 17.86 ml/l/h. Meanwhile, the power consumption by combining SEEOFP and TL was about 37.1% lower than using TL alone. This study demonstrates that using optimal light sources and proper dilution of dark fermentation effluent, the performance of photo-H₂ production can be markedly enhanced along with a reduction of power consumption.     

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.     

Impairment of NADH dehydrogenase for increased hydrogen production and its effect on metabolic flux redistribution in wild strain and mutants of Enterobacter aerogenes

An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H₂/mol glucose, respectively, while it was 1.16 mol H₂/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H₂ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.     

Accumulation of O2-tolerant phenotypes in H2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H₂-evolving activities of up to 10 times the O₂-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H₂-metabolism clones following exposure to increasing amounts of O₂, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H₂ following exposure to O₂ concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H₂-production activity once O₂ is removed. These results suggest that O₂-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O₂ when it is actively catalyzing H₂ evolution.     

Metabolic flux analysis of hydrogen production network by Clostridium butyricum W5: Effect of pH and glucose concentrations

Fermentative hydrogen production by strict anaerobes has been widely reported. There is a lack of information related to metabolic flux distribution and its variation with respect to fermentation conditions in the metabolic production system. This study aimed to get a better understanding of the metabolic network and to conduct metabolic flux analysis (MFA) of fermentative hydrogen production by a recently isolated Clostridium butyricum strain W5. We chose the specific growth rate as the objective function and used specific H₂ production rate as the criterion to evaluate the experimental results with the in silico MFA. For the first time, we constructed an in silico metabolic flux model for the anaerobic glucose metabolism of C. butyricum W5 with assistance of a modeling program MetaFluxNet. The model was used to evaluate metabolic flux distribution in the fermentative hydrogen production network, and to study the fractional flux response to variations in initial glucose concentration and operational pH. The MFA results suggested that pH has a more significant effect on hydrogen production yield compared to the glucose concentration. The MFA is a useful tool to provide valuable information for optimization and design of the fermentative hydrogen production process.     

Hydrogen production from acid hydrolyzed molasses by the hydrogen overproducing Escherichia coli strain HD701 and subsequent use of the waste bacterial biomass for biosorption of Cd(II) and Zn(II)

This study was devoted to investigate production of hydrogen gas from acid hydrolyzed molasses by Escherichia coli HD701 and to explore the possible use of the waste bacterial biomass in biosorption technology. In variable substrate concentration experiments (1, 2.5, 5, 10 and 15 g L⁻¹), the highest cumulative hydrogen gas (570 ml H₂ L⁻¹) and formation rate (19 ml H₂ h⁻¹ L⁻¹) were obtained from 10 g L⁻¹ reducing sugars. However, the highest yield (132 ml H₂ g⁻¹ reducing sugars) was obtained at a moderate hydrogen formation rate (11 ml H₂ h⁻¹ L⁻¹) from 2.5 g L⁻¹ reducing sugars. Subsequent to H₂ production, the waste E. coli biomass was collected and its biosorption efficiency for Cd²⁺ and Zn²⁺ was investigated. The biosorption kinetics of both heavy metals fitted well with the pseudo second-order kinetic model. Based on the Langmuir biosorption isotherm, the maximum biosorption capacities (q_max) of E. coli waste biomass for Cd²⁺ and Zn²⁺ were 162.1 and 137.9 (mg/g), respectively. These q_max values are higher than those of many other previously studied biosorbents and were around three times more than that of aerobically grown E. coli. The FTIR spectra showed an appearance of strong peaks for the amine groups and an increase in the intensity of many other functional groups in the waste biomass of E. coli after hydrogen production in comparison to that of aerobically grown E. coli which explain the higher biosorption capacity for Cd²⁺ or Zn²⁺ by the waste biomass of E. coli after hydrogen production. These results indicate that E. coli waste biomass after hydrogen production can be efficiently used in biosorption technology. Interlinking such biotechnologies is potentially possible in future applications to reduce the cost of the biosorption technology and duplicate the benefits of biological H₂ production technology.     

Solar photocatalysis for detoxification and disinfection of water: Different types of suspended and fixed TiO2 catalysts study

Photocatalysis by titanium dioxide (TiO₂), operational in the UV-A domain with a potential use of solar radiation, could be an alternative to conventional water detoxification and disinfection technologies. However, employing the photocatalyst as a suspension or slurry makes the scaling-up of the process difficult, as the TiO₂ has to be removed from the decontaminated water to be reused several times. In this work the photocatalytic activity of different types of TiO₂ catalyst (Degussa P-25, Millennium PC-100 and PC-500, Tayca AMT-100 and AMT-600) in suspension or coated on fibrous web were studied in both decontamination and disinfection experiments at laboratory scale. Gallic acid was chosen as the model pollutant for detoxification experiments and Escherichia coli as the model microorganism for disinfection experiments. The influence of the surface area and other characteristics of TiO₂ are discussed concerning the photocatalytic properties of TiO₂. The role of adsorption is suggested, indicating that the reaction occurs at the TiO₂ surface and not in the solution. Gallic acid degradation kinetics were found to be of the same extent for both TiO₂ suspended and fixed, whereas for the bacterial inactivation efficiency was significantly less important with coated than with suspended TiO₂.