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 from a carbon-monoxide oxidation pathway in Rubrivivax gelatinosus

Upon feeding CO to the gas phase of a photosynthetic bacterium Rubrivivax gelatinosus CBS, a CO oxidation: H₂ production pathway is quickly induced. Hydrogen is produced according to the equation CO+H₂O→CO₂+H₂. Two enzymes are known to be involved in this pathway: a CO dehydrogenase (CODH) with a pH optimum of 8.0 and above, and a hydrogenase with a pH optimum near 7.5. Carbon monoxide dehydrogenase also displays a temperature optimum near 50°C. When CO mass transfer is not limited during a CO uptake measurement, an extreme fast rate of CO uptake was determined, allowing for the removal of near 87% of the dissolved CO from a bacterial suspension within 10 s. This process has therefore two potential applications, one in the production of H₂ gas as a clean renewable fuel using the linked CO oxidation: H₂ production pathway, and another in using the CODH enzyme itself as a fuel–gas conditioning catalyst. These applications thereby will improve the overall H₂ economy when gasified waste biomass serves as the inexpensive feedstock.     

Development of natural seawater-based continuous biohydrogen production process using the hyperthermophilic archaeon Thermococcus onnurineus NA1

Previously, we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 could produce hydrogen from various resources including carbon monoxide (CO) with high productivity. In this study, a high-performance and cost-effective fermentation process was developed as a prerequisite for the industrial implementation of H₂ production from CO using the strain. Firstly, an economical medium was formulated on the basis of natural seawater. Cysteine as a reducing agent was found to be an important factor to consider. Secondly, three variables, initial concentration of yeast extract, CO supply rate and agitation rate, were statistically optimized in batch culture by applying a response surface methodology. Notably, biomass productivity of 0.39 g/L/h could be observed and cell density reached 14 optical units at 600 nm, which is exceptionally high among members of the order Thermococcales. Finally, continuous cultures were tested at various dilution rates with new medium formulation and optimized culture conditions. As a result, the steady state of H₂ production and cell growth were stably maintained, yielding H₂ production rate of 471.5 mmol/L/h at a dilution rate of 0.1 h^?1, which is one of the highest values at ambient CO pressure to date. Our study shows that T. onnurineus NA1 has outstanding potential as an economical and high performance industrial strain suitable for long-term processes for CO-dependent H₂ production.     

Methane free biohydrogen production from carbon monoxide using a continuously operated moving bed biofilm reactor

Biological water-gas shift (WGS) reaction is a green and sustainable alternative to thermochemical-catalytic WGS process for hydrogen production from carbon monoxide (CO). However, CO tolerant carboxydotrophic microbes for hydrogen production and scaling up the technology using a bioreactor system present challenges in successful application of this technology. This study demonstrated the capability of anaerobic microbial consortium for biohydrogen production from CO using a moving bed biofilm reactor (MBBR). The CO conversion pathway followed by the anaerobic biomass was first elucidated by inhibiting the methanogens present using 2-bromoethanesulfonate (BES) at an optimum concentration of 10 mmol/L. An increase in inlet CO concentration to the MBBR enhanced the H₂ production, but the CO conversion efficiency was low. More than 80% CO conversion efficiency was obtained only for a low inlet CO concentration. A maximum H₂ concentration of 19.5 mmol/L along with 2 mmol/L of acetate were obtained for 36 mmol/L of inlet CO concentration in the bioreactor. The carbon flux analysis showed that the CO was mainly utilized for methane free H₂ production, and only <10% of carbon flux was diverted towards acetate formation. Overall, this study demonstrated that MBBR system can be used for steady state biohydrogen production over a prolonged operation period.     

Steam reforming of n-heptane for production of hydrogen and syngas

Simultaneous production of hydrogen and syngas from the catalytic reforming of n-heptane in circulating fast fluidized bed reactors (CFFBR) and circulating fast fluidized bed membrane reactors (CFFBMR) is investigated. This paper presents modeling and simulation approach for the analysis of these reformers. Complete conversion of heptane (100%) is attained at high steam to carbon feed ratios and shorter reactor lengths by both configurations. However, the CFFBMR is very efficient in hydrogen production and can produce exit hydrogen yield up to 473.14% higher than the CFFBR. It was found that operating the CFFBMR at the optimal conditions results in a minimum value of hydrogen to carbon monoxide ratio (H₂/CO) within the recommended practical range for the syngas used as a feedstock for the gas to liquid processes (GTL). The results of the sensitivity analysis conducted for the CFFBMR has shown that the reaction side pressure and the feed temperature have significant effects on increasing the heptane conversion (up to 100%) and the temperature effect is stronger than the reaction side pressure effect. Considerable improvement in the hydrogen to carbon monoxide ratio (H₂/CO) has been achieved by increasing the reaction side pressure, while the high feed temperature has negative effect on this ratio. It seems that the practical range of H₂/CO ratio can be achieved by controlling the reformer length and the right combinations of the operating conditions.     

The effect of reduction of pretreated NiO–ZnO catalysts on the water–gas shift reaction for hydrogen production as studied by in situ DRIFTS/MS

The water–gas shift (WGS) reaction on co-precipitated NiO–ZnO catalysts at different reduction temperatures has been studied by a temperature-programmed reaction using in situ diffuse reflectance infrared Fourier Transform Spectroscopy, coupled with mass spectroscopic (in situ DRIFTS/MS) techniques. The results reveal that a catalyst reduced at 493 K (labeled H220) showed higher activity than one reduced at 673 K (labeled H400) due to the ability of NiO on the H220 catalyst to promote CO conversion of the WGS reaction. In situ DRIFTS/MS studies show that there are three adsorbed species over the H220 catalyst at room temperature: adsorbed CO bands, molecularly adsorbed H₂O and carboxyl species. Increasing the temperature to 423 K led to the emergence of CO₂ and H₂ and the disappearance of carboxyl species. However, the low catalytic activity of the H400 catalyst could be attributed to the conversion of the NiO sites to reduced Ni metal sites, which (i) adsorbed CO as the strong linearly bonded CO on the catalyst surface, slowing down the CO reaction, and (ii) showed a lower H₂O uptake.     

Fermentative hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture

In this study, production of hydrogen (H₂) from glucose, xylose, galactose, mannose, arabinose and rhamnose by a strain isolated from activated sludge was investigated. The strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter amalonaticus strain SA01 (99%). Optimal cultivation parameters for H₂ production and growth such as pH and temperature were investigated. H₂ yields from glucose at optimal conditions (pH 6.0 and 34 °C) were 1.82 ± 0.02 mol-H₂/mol-glucose. Strain CMC-1 fermented galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation period lowered residual formate level in the media and improved H₂ production for galactose, mannose and xylose (1.68 ± 0.24, 1.93 ± 0.14 and 1.63 ± 0.07 mol-H₂/mol-substrate respectively).     

The mechanism characterizations of methane steam reforming under coupling condition of temperature and ratio of steam to carbon

To elucidate the coupling effects of temperature and ratio of steam to carbon on the methane steam reforming process, the characterizations of methane steam reforming at different temperature and ratio of steam to carbon in term of distribution of H₂ and CO, and the elementary reaction rate were investigated. Meanwhile, the formation mechanisms of H₂ and CO via sensitivity analysis and reaction path analysis were obtained. The results showed that the coupling effects of temperature and ratio of steam to carbon on the methane steam reforming were higher than that of individual factor. The effects of temperature on the methane steam reforming were higher than that of the ratio of steam to carbon. The adsorption and desorption reaction of CH₄ on the surface of Ni-based catalyst had the most obvious effect on the sensitivity of H₂, CH₄ and CO. Besides, the effects of adsorption and desorption reaction of H₂O on the sensitivity of H₂ were higher than that of CH₄ and CO. Hydrogen was generated by the desorption reaction of H(s) in the adsorbed state and from three generating paths: a) CH₄(s) dissociated directly or reacted with O(s) to form H(s); b) The dissociation reaction of H₂O(s) produced H(s); c) OH(s) dissociated directly or reacted with C(s) to form H(s). Carbon monoxide was generated from single path: CH₄(s)→CH₃(s)→CH₂(s)→CH(s)→C(s)→CO(s)→CO(g).     

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.     

Preparation of mesoporous nanocrystalline CuO–ZnO–Al2O3 catalysts for the H2 purification using catalytic preferential oxidation of CO (CO-PROX)

In this article, CuO–ZnO–Al₂O₃ catalysts with various copper contents were synthesized by a co-precipitation method and employed for the elimination of carbon monoxide from a mixture of 97% H₂, 1% CO and 2% O₂ at atmospheric pressure via carbon monoxide preferential oxidation (CO-PROX). The influence of the copper and zinc contents on the physicochemical characteristics and catalytic performance was investigated. The prepared samples were characterized using the N₂ adsorption-desorption (BET), X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM and SEM) and temperature programmed reduction (TPR) techniques. The increment in CuO loading improved the activity of CuO–ZnO–Al₂O₃ catalysts for CO oxidation reaction. Among the prepared catalysts, the 50%CuO-3% ZnO-47% Al₂O₃ catalyst calcined at 400 °C with a BET area of 82.3 m²/g exhibited the best activity with a CO conversion of 88.9% at 125 °C. The effects of the presence of CO₂ and H₂O in the reaction feed stream and gas hourly space velocity (GHSV) were also studied.     

Effect of nickel loading on hydrogen production and chemical oxygen demand (COD) destruction from glucose oxidation and gasification in supercritical water

Gasification and partial oxidation of 0.25 molar glucose solution was conducted over different metallic nickel (Ni) loadings (7.5, 11, and 18 wt%) on different catalyst supports (θ-Al₂O₃ and γ-Al₂O₃) in supercritical water. Experiments were carried out at three different temperatures (T) of 400, 450, and 500 °C at constant pressure of 28 MPa and a 30 min reaction time (t). For comparison, some experiments were conducted using high loading commercial catalyst (65 wt% Ni on Silica–alumina). Hydrogen peroxide (H₂O₂) was used as a source of oxygen in the partial oxidation experiments. Oxygen to carbon molar ratios (MR) of 0.5–0.9 were examined to increase the hydrogen production via carbon monoxide (CO) production. Results showed that in the absence of the catalyst, the optimum molar ratio was 0.8 i.e. 80% of the amount of oxygen required for complete oxidation of glucose. At a molar ratio of 0.8, the hydrogen yield was 0.3 mol/mol, as compared to 0.2 mol/mol glucose at molar ratio of 0.5 and 0.9. This optimized oxygen dose was adopted as a base line for catalysts evaluation. The main gaseous products were carbon dioxide (CO₂), carbon monoxide (CO), hydrogen (H₂), and methane (CH₄). Results also showed that the presence of Ni increased the total gas yield increased in the 7.5–18 wt Ni/Al₂O₃ catalyst. An increase in MR from 0.55 to 0.8 increased the of carbon dioxide and hydrogen yields from 1.8 to 3.8 mol/mol glucose and from 0.9 to 1.1 mol/mol. The carbon monoxide and methane yields remain constant at 2 and 0.5 mol/mol glucose, respectively. The introduction of hydrogen peroxide (H₂O₂) prior to the feed injection inhibited the catalyst activity and did not increase the hydrogen yield whereas the introduction of H₂O₂ after 15 min of reaction time increased the hydrogen yield from 0.62 mol/mol to 1.5 mol/mol. This study showed that approximately the same hydrogen yield can be obtained from the synthesized low nickel alumina loading (18 wt%) catalyst as with the 65 wt% nickel on silica–alumina loading commercial catalyst. The highest H₂ yield of 1.5 mol/mol glucose was obtained with commercial Ni/silica–alumina with a BET surface area of 190 m²/g compared to 1.2 mol/mol with the synthesized Ni/θ alumina with a BET surface area of 46 m²/g.     

A novel Rh–Ir electrocatalyst for the oxygen reduction reaction and the hydrogen and methanol oxidation reactions

A novel Rh–Ir based material was synthesized by pyrolysis of an Ir₄(CO)₁₂/Rh₆(CO)₁₆ mixture in a reductive (H₂) atmosphere. The material was characterized by FTIR spectroscopy, X-ray diffraction, energy dispersive spectroscopy and scanning electron microscopy, and was evaluated as electrocatalyst for oxygen reduction and hydrogen and methanol oxidation by rotating disk electrode measurements. The bimetallic material shows a high catalytic activity for the oxygen reduction reaction and is also capable to carry out the hydrogen oxidation reaction even in the presence of carbon monoxide in different concentrations (100 ppm and 0.5%), in contrast with commercial platinum catalysts, which become easily deactivated by CO. The activity of the catalyst for methanol oxidation is acceptable but still low in comparison with Pt–Ru. The results show that the new bimetallic catalyst is a potential candidate to be evaluated as both cathode and anode in a reforming hydrogen PEMFC, and as an anode in a direct methanol fuel cell.     

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 production from C1 compounds by a novel marine hyperthermophilic archaeon Thermococcus onnurineus NA1

A novel marine hyperthermophile, Thermococcus onnurineus NA1, was found to grow on C1 carbon compounds, such as formate and carbon monoxide (CO), and produce hydrogen (H₂). In the present study, the growth and H₂ production of NA1 were examined to determine its potential as H₂ producer. NA1 showed relatively high specific growth rates, 0.48 h⁻¹ and 0.40 h⁻¹ with CO (20%, v/v) and formate (100 mM), respectively, when cultivated in batch mode in a minimal salt medium fortified with 1.0 g L⁻¹ yeast extract. On the other hand, cell growth in both cases stopped at approximately 6 h and the final cell densities were extremely low at 18.2 and 12.1 mg protein L⁻¹ with CO and formate, respectively. The maximum final cell density could be improved greatly to 36.0 mg protein L⁻¹ by optimizing CO content (50%, v/v) and yeast extract concentration (4.0 g L⁻¹), but it was still very low. During the cell growth, formate and CO were used as energy source rather than carbon source. In the resting cell experiments, NA1 exhibited remarkably high H₂ production activities as 385.0 and 207.5 μmol mg protein⁻¹ h⁻¹ for CO and formate, respectively. When formate (100 mM) or CO (100%, v/v) was added repeatedly at 30–35 h intervals, NA1 showed consistent H₂ production for 3 cycles with a yield of approximately 1.0 mol H₂ mol⁻¹ for both CO and formate. This study suggests that T. onnurineus NA1 has a high H₂ production potential from formate or CO but a method for achieving a high cell density culture is needed.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Biohydrogen by Rhodobacter sphaeroides during photo-fermentation: Mixed vs. sole carbon sources enhance bacterial growth and H2 production

Mixed carbon sources fermentation by bacteria is a promising approach for biohydrogen (H₂) production biotechnology. In the present study, growth and Н₂ production by purple bacteria Rhodobacter sphaeroides MDC6521 during mixed carbon sources (succinate + acetate, succinate + malate, and malate + acetate) photo-fermentation was investigated. The growth rate of bacteria in mixed carbon sources containing medium was of ∼0.33 h⁻¹ which was considerably higher (1.3–1.7-fold) compared with sole carbon substrate containing one. Moreover, the H₂ production during photo-fermentation of succinate and acetate mixture was of ∼6.5 mmol H₂ g⁻¹ (dry weight of biomass) and significantly more (∼2–3-fold) than that with appropriate sole sources and higher (1.5-fold) than that with succinate and malate mixture. Probably, supplementation of the mixed carbon sources into bacterial culture alters the mode of metabolism, resulting in enhanced H₂ production, thus they can be preferable compared to the sole carbon source. The changed F_OF₁-ATPase activity of membrane vesicles suggested its important role in the increase of Н₂ production efficiency. The results showed that mixed carbon sources provide more H₂ than the sole carbon substrates and succinate with acetate mixture is better than succinate with malate.     

Growth characteristics and hydrogen production by Rhodobacter sphaeroides using various amino acids as nitrogen sources and their combinations with carbon sources

Some amino acids (alanine, asparagine, glutamate, glycine, proline, and tyrosine) were used as nitrogen sources in combination with carbon sources (succinate and malate) to study growth properties and H₂ production by purple non-sulfur bacterium Rhodobacter sphaeroides strains A-10 and D-3. Both strains produced H₂ in succinate–glutamate and malate–glutamate media. Succinate was a better carbon source than malate. In comparison with strain D-3, strain A-10 was able to utilize proline, alanine or tyrosine as nitrogen sources in succinate medium and to produce H₂. Both strains were unable to produce H₂ in the presence of asparagine or glycine as nitrogen sources. N,N′-dicyclohexylcarbodiimide, the F₀F₁-ATPase inhibitor, led to marked inhibition of H₂ production activity of R. sphaeroides. The results suggest that the R. sphaeroides cells growth can be achieved by the use of a large diversity of substrates but only some of them can increase the H₂ production rate.     

Effect of different gasifying agents (steam,H2O2, oxygen,CO2, and air) on gasification parameters

Two sensitivity analyses were performed in an Aspen simulation of fluidized bed gasification for five different gasifying agents such as steam, hydrogen peroxide (H₂O₂), pure oxygen (O₂), carbon dioxide (CO₂), and air. In the first sensitivity analysis, the modified equivalence ratio (MER) was varied (0.22-0.36). For the varied modified equivalence ratio (MER), %hydrogen, H₂/CO molar ratio, and hydrogen yield were the highest in steam-gasification, but %carbon monoxide, %methane, CO yield, and the lower heating values (LHV) were the highest in CO₂-gasification. In the second sensitivity analysis, the freeboard temperature was varied (500-900 °C). With increasing freeboard temperature, %hydrogen and %carbon monoxide increased while %carbon dioxide and %methane decreased for all the gasifying agents. Also, with increasing freeboard temperature, the LHV decreased and the hydrogen yield, CO yield, and the gas production rate increased for all the gasifying agents, but the H₂/CO molar ratio increased only in oxygen, air, and CO₂-gasification.     

Tri-metallic Ni–Co modified reducible TiO2 nanocomposite for boosting H2 production through steam reforming of phenol

Well-designed Co₃O₄ nanocubes (NCs) dispersed NiO/TiO₂ to construct tri-metallic reducible NiO/TiO₂/Co₃O₄ NCs structured catalyst for steam-reforming of phenol (SRP) with enhanced hydrogen production has been investigated. The controlled morphology with good dispersion was obtained, enabling efficient SRP toward selective H₂ production. Using 10% NiO- 5% Co₃O₄ NCs/TiO₂ composite, H₂ yield of 69.91% and phenol conversion of 78.4% was achieved, significantly higher than using NiO/TiO₂ and TiO₂ samples. The cubical structured Co₃O₄ dispersed NiO/TiO₂ composite showed significantly improved H₂ yield and phenol conversion due to strong metal-support interaction with reducible support for providing more active sites. The H₂ production was further increased by increasing reaction temperature, phenol concentration, feed flow rate and catalysts loading, however, they have adverse effect on the selectivity due to more CO formation. The composite catalyst possesses excellent activity and stability due to strong tri-metallic interaction and exceptional electronic interfaces. The spent catalyst analysis confirms the formation of graphene and carbon nanotubes over the reducible support. This study reveals that Co₃O₄ NCs are able to increase NiO/TiO₂ activity for H₂ production by inhibiting carbon monoxide formation and would be beneficial in other reforming applications.