Metabolic flux analysis of biological hydrogen production by Escherichia coli

For hydrogen to be a viable energy carrier, it is essential to generate it from renewable sources. A promising route is hydrogen generation from biological methods. However, the low yield of hydrogen is a constraint. Significant improvement in the yield is expected through genetic modification of the microorganism. Metabolic flux analysis (MFA) can be used to analyze the effect of genetic modification on the yield a priori.In this paper MFA has been applied to hydrogen production using growth of Escherichia coli on glucose. Flux analysis was carried out on experimental data from literature. Hydrogen production using parent strain was 0.17 mol per mole of glucose consumed. It increased to 0.23 mol for the strain lacking lactate dehydrogenase. MFA was also used to determine the feasible operating space for hydrogen production against the varying yields of other metabolites. It was found that production of ethanol and acetate is necessary for hydrogen production, while production of succinate and lactate is not necessary.     

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.     

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.     

Characterization and optimization of hydrogen photoproduction by a saltwater blue-green alga, Oscillatoria sp. Miami BG7. I. Enhancement through limiting the supply of nitrogen nutrients

Hydrogen photoproduction by a marine, non-heterocystous, filamentous blue-green alga, Oscillatoria sp. Miami BG7 was studied in its relationship to nitrogen nutrients in the culture medium. When a combined nitrogen sufficient culture was inoculated into a combined nitrogen limited medium, cellular content of chlorophyll and protein decreased whereas the carbohydrate content increased significantly during the culture period. Accompanying this change of cellular composition the oxygen photoproduction capability decreased and hydrogen photoproduction capability increased dramatically. The maximum rate of hydrogen photoproduction was 260 μmol/mg chlorophyll/h. Hydrogen production was strictly light dependent under an anaerobic condition. Along with hydrogen photoproduction, carbon dioxide was also produced. The ratio of hydrogen and carbon dioxide photoproduction was approximately two. The mechanism of hydrogen photoproduction enhancement in combined nitrogen limited culture is apparently related to the increase of nitrogenase synthesis, decline of photosystem II activity, and the accumulation of electron donor substances during culture. Another interesting feature of hydrogen production was accumulation of gas in the flasks without any special treatments. This is related to the lack of hydrogen uptake activity in this strain. These results indicate that this strain has favorable characteristics for future application to biosolar fuel production.     

Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii

Sulfur deprivation, which is considered as an efficient way to trigger long-term hydrogen photoproduction in unicellular green algae has two major effects: a decrease in PSII which allows anaerobiosis to be reached and carbohydrate (starch) storage. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in the green alga Chlamydomonas reinhardtii, little remains known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify those steps likely to limit the starch-dependent hydrogen production, we have designed a genetic screening procedure aimed at isolating mutants of the green alga C. reinhardtii affected in starch mobilization. Using two different screening protocols, the first one based on aerobic starch degradation in the dark and the second one on anaerobic starch degradation in the light, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std1-8) with the first screen and ten (sda1-10) with the second. Most of the mutant strains isolated in this study showed a reduction or a delay in the PSII-independent hydrogen production. Further characterization of these mutants should allow the identification of molecular determinants of starch-dependent hydrogen production and supply targets for future biotechnological improvements.     

The microalga Chlamydomonas reinhardtii CW-15 as a solar cell for hydrogen peroxide photoproduction: Comparison between free and immobilized cells and thylakoids for energy conversion efficiency

Immobilized cells and thylakoid vesicles of the microalga Chlamydomonas reinhardtii CW-15 have been developed as a solar cell because of their capabilities of producing hydrogen peroxide. This compound is an efficient and clean fuel used for rocket propulsion, motors and for heating. Hydrogen peroxide is produced by the photosystem in a catalyst cycle in which a redox mediator (methyl viologen) is reduced by electrons obtained from water by the photosynthetic apparatus of the microalga and it is re-oxidized by the oxygen dissolved in the solution. The photoproduction has been investigated using a discontinuous system with whole cells, or thylakoid vesicles, free or immobilized on alginate. The stimulation by azide as an inhibitor of catalase has also been analyzed. Under determined optimum conditions, the photoproduction by Ca-alginate entrapped cells, with a rate of 33 μmol H₂O₂/mg Chl.h, was maintained for several hours with an energy conversion efficiency of 0.25%.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

Effect of inactivation of genes involved in ammonium regulation on the biohydrogen production of Rhodobacter capsulatus

Hydrogen production by nitrogenase is an energetically expensive process for the cell, hence strictly controlled at different levels. Ammonium is one of the substances regulating nitrogenase activity. The key proteins in the regulation of nitrogenase by ammonium are two regulatory proteins; GlnB and GlnK. In order to increase hydrogen production of Rhodobacter capsulatus DSM1710 (wild type strain) grown on agricultural materials/wastes, ammonium inhibition of nitrogenase enzyme has to be eliminated. In this study, GlnB and GlnK were targeted to be inactivated by in frame site-directed mutagenesis. The glnB mutant R. capsulatus (GP1 strain) was obtained at the end of mutagenesis studies. In the case of glnK, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained.The effect of ammonium on the growth and hydrogen production of R. capsulatus GP1 was investigated and compared with DSM1710. Both DSM1710 and GP1 strains were effectively utilized acetate. The mutation did not affect cell growth significantly at different ammonium levels. Ammonium negatively affected hydrogen production of GP1 strain as well as the DMS1710. However, hydrogen production was significantly low in GP1 strain. The ammonium inhibition of hydrogen production could not be removed in glnB mutant probably due to the presence of an active GlnK protein in the cell. Therefore, GlnK has much more important role in the ammonium dependent control of nitrogenase than GlnB does. The growth and hydrogen production kinetics of R. capsulatus DSM1710 and GP1 were modelled. They were shown to fit to Logistic Model and Modified Gompertz Model, respectively.     

Solar hydrogen production and its development in China

Because of the needs of sustainable development of the mankind society and natural environment building a renewable energy system is one of the most critical issues that today's society must address. In the new energy system there is a requirement for a renewable fuel to replace current energy carrier. Hydrogen is an ideal secondary energy. Using solar energy to produce hydrogen in large scale can solve the problems of sustainability, environmental emissions, and energy security and become the focus of the international society in the area of energy science and technology. It has also been set as an important research direction by many international hydrogen programs. The Ministry of Science and Technology of China supported and launched a project of National Basic Research Program of China (973 Program) – the Basic Research of Mass Hydrogen Production using Solar Energy in 2003 for R&D in the areas of solar hydrogen production. The current status of solar hydrogen production research is reviewed and some significant results achieved in the project are reported in this paper. The trends of development and the future research directions in the field of solar hydrogen production in China are also briefly discussed.     

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.     

Review on effective parameters in electrochemical hydrogen storage

Today, energy has become one of the most important concerns of developing countries. The use of non-renewable energy sources, as well as the production of pollution, has led to growing efforts to replace fossil fuels, which are the most important energy sources in the modern world. Hydrogen as a clean fuel has attracted a lot of attention in recent years. Various methods have been reported for the production and storage of hydrogen. According to their advantages and disadvantages, it can be said that electrochemical hydrogen storage method is superior to other methods in terms of cost, safety, and optimum condition. The electrochemical hydrogen storage is done in a variety of techniques, and in recent years, the chronopotentiometry method has become one of the most popular methods for scientists. In chronopotentiometry technique, several parameters such as the reference electrode, the counter electrode, the working electrode, electrolyte, and current density are important. In this review, we investigated the articles that have been done in this regard from 2000 to 2020. This review can help scientists to better understand the electrochemical hydrogen storage system.     

Performance analysis and economic competiveness of 3 different PV technologies for hydrogen production under the impact of arid climatic conditions of Morocco

For green hydrogen production, the choice of the appropriate renewable energy source to drive the water electrolysis process is crucial. Currently, solar Photovoltaic (PV) energy is one of the most popular and cheapest renewable energy sources; however, the performance of this technology is highly affected by the weather condition especially after the exposition to harsh climate conditions for long periods. Accordingly, the aim of this study is to assess the appropriate PV technology for hydrogen production under the impact of arid climatic conditions. For this reason, we evaluated the hydrogen production from 3 PV technologies, namely: monocrystalline (m-Si), polycrystalline (p-Si) and amorphous (a-Si) technologies exposed outdoors for a period of 3 years under the arid climatic conditions of Errachidia, Morocco. In addition, the degradation rate of each technology has been calculated and its impact on hydrogen production and its cost has been investigated.The results show that, the technology with the higher yearly hydrogen yield is the p-Si with 37.07 kg/kWp, followed by the m-Si with 36.84 kg/kWp and finally the a-Si with 36.68 kg/kWp. As for the cost of hydrogen production, the lowest cost was found in the case of the p-Si technology as well with 4.89 $/kg, whereas for the m-Si and a-Si technologies it was found equal to 5.48 $/kg and 6.28 $/kg respectively. However, the evaluation the impact of the PV modules degradation reveals that p-Si is technology affect the most with an annual degradation rate of 0.92%, followed by the a-Si with 0.72% and m-Si technologies with 0.45%. Nonetheless, when taken in consideration the impact of the degradation on the cost of hydrogen production, the p-Si remain the most cost effective technology even though the cost has increase to 5.32 $/kg, 5.78 $/kg and 6.67 $/kg for the p-Si, m-Si and a-Si technologies respectively.     

Photoproduction of hydrogen by dye-sensitized systems

Visible light-induced production of hydrogen has been investigated in five different systems. These are: safranine O/EDTA, safranine T/EDTA, proflavine/EDTA, acridine orange/EDTA, and acridine yellow/EDTA, with and without added K₂PtCl₆. In the two safranine systems photoproduction of hydrogen was observed even in the absence of a Pt catalyst. Also, the addition of an electron mediator such as methyl viologen was found not necessary. Acridine yellow/EDTA/K₂PtCl₆ has been shown to be the best system examined, which upon addition of Triton X-100, showed further enhancement of the rate of hydrogen evolution.     

Techno-economic assessment of renewable hydrogen production and the influence of grid participation

Large-scale hydrogen production facilities will be required to supply the chemical energy demand of certain industries in the future. The case for such production plants based on individual adapted PV and wind farms has been addressed in several studies. However, most studies focus on an island solution of the evaluated plant and therefore, do not allow grid assistance which significantly reduce the installed capacity of the corresponding units. To address this issue, we developed a tool with a linear programming approach to evaluate any location around the world for its renewable hydrogen production costs and the influence on the plant layout depending on its interaction with the grid. A detailed techno-economic evaluation has been performed for five locations where hydrogen production costs in the range of 4–6 €₂₀₂₀/kg have been retrieved. Furthermore, it is shown that with perspective cost data the costs can further be reduced to 2.50 €₂₀₂₀/kg.     

An evaluative report and challenges for fermentative biohydrogen production

Hydrogen, the most abundant and lightest element in the universe, has enormous potential as a future energy. High conversion efficiency, recyclability and nonpolluting nature of hydrogen make it the fuel of future. Various microorganisms are explored for producing hydrogen by exploiting variety of biological organic substrates. The target is the genetic improvement of the organism or the biochemical pathway required for biohydrogen production and devising path even better in comparison to the other production methods.The present review discusses different methods of biohydrogen production specifically by the fermentative route, physical factors affecting its production and other aspects for enhancement in the yield of hydrogen production. Metabolic engineering strategies for enhancement in hydrogen production to overcome different limitation have been also summarized.     

Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures

Unsynchronized Chlamydomonas reinhardtii cells subsequently deprived of sulfur produce H₂ under continuous illumination in the laboratory for 3–4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronize their growth and cell division. While it is clear that only insignificant amounts of H₂ can be produced by sulfur-deprived cells during the night period, little work has been done to examine the effects of the light/dark cycles preceding sulfur deprivation on subsequent H₂ photoproduction. We show that (a) C. reinhardtii cells exhibit synchronized growth and cell division in the presence of acetate, (b) cells with the highest specific rates of H₂ photoproduction also have the highest rates of biomass accumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetate accumulation, but not with H₂ photoproduction. This work shows that it is possible to maximize the production of H₂ by sulfur-depriving synchronized cultures at about 4 h after the beginning of the light period.     

Effect of carbon coating on Cu electrodes for hydrogen production by water splitting

In recent years, fossil fuel depletion has been increasing, which leads to environmental issues. Hydrogen energy is considered a promising renewable energy to replace fossil fuels because it is a sustainable, clean, and green energy source. Among hydrogen production methods, water splitting has the highest reliability and is used the most often. Platinum is normally used as water splitting catalyst and an electrode. However, there has been much effort to replace it as such owing to its high cost. Copper (Cu) is not used as water splitting catalyst or an electrode, despite its high current density, because of its corrosive properties. In this study, carbon was coated onto a Cu substrate and a hydrogen production experiment was carried out with 0.1 M Na₂SO₄ and 0.1 M H₂SO₄ electrolytes. As a result, the carbon coating decreased oxidation rate of the Cu electrode and effected stability in short-term hydrogen evolution experiment. This indicates the possibility of carbon-Cu electrode with other catalytic materials.     

Hydrogen production from alcohols and ethers via cold plasma: A review

Hydrogen is one of the most promising energy and the fuel for fuel-cell-powered automobiles. Liquid fuels have been considered as the most suitable source for onboard hydrogen production, and cold plasma is proved to be a potential way to convert them. In this review, the conversion of methanol, ethanol and dimethyl ether (DME) using different types of cold plasma has been summarized. Hydrogen is the main product with different by-products depending on reaction conditions. The conversion of liquid fuels for hydrogen production via cold plasma has good prospects, and reaction conditions optimization and reactor design are very important for its future application onboard.     

The future of hydrogen energy: Bio-hydrogen production technology

The widespread use of non-renewable energy has caused serious environmental problems such as global warming and the depletion of fossil fuels. Hydrogen, as a well-known carbon-free gaseous fuel, has become the most promising energy carrier for future energy. Hydrogen has an excellent mass-basis calorific value and no carbon atom contained, which makes it to be an attractive fuel for various power devices (like the internal combustion engine, gas turbine, and fuel cell). Nowadays, the production of hydrogen is still predominated by fossil-based techniques, which is considered undesirable due to low conversion efficiency and release of greenhouse gases. It is necessary to find green and sustainable hydrogen production routes with low energy consumption and cost. In this paper, the different hydrogen production technologies via fossil routes or non-fossil routes are reviewed in general, and it is found that bio-hydrogen production has certain environmental advantages and broad prospects compared with other hydrogen production technologies. Then, the characteristics and research status of different bio-hydrogen production technologies are discussed in depth. It is found that each bio-hydrogen production technique has its own advantages, challenges, and applicability. The economic analysis of bio-hydrogen energy is also performed from the aspects of production, storage, and transportation. The results show that bio-hydrogen production technology could be a good possibility way for producing renewable hydrogen, which is of high efficiency and thus competitive over other hydrogen production methods both in economics and environmental benefits.