Statistical optimization of fermentative hydrogen production from xylose by newly isolated Enterobacter sp. CN1

Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett–Burman design, xylose, FeSO₄ and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box–Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO₄ 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 ± 65 ml H₂/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H₂/L xylose medium, 1102 ml H₂/L glucose medium and 977 ml H₂/L sucrose medium; the maximum hydrogen yield were 2.0 ± 0.05 mol H₂/mol xylose, 0.64 mol H₂/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose.     

Design of a novel flat-plate photobioreactor system for green algal hydrogen production

Some green microalgae have the ability to harness sunlight to photosynthetically produce molecular hydrogen from water. This renewable, carbon-neutral process has the additional benefit of sequestering carbon dioxide and accumulating biomass during the algal growth phase. We document the details of a novel one-litre vertical flat-plate photobioreactor that has been designed to facilitate green algal hydrogen production at the laboratory scale. Coherent, non-heating illumination is provided by a panel of cool-white light-emitting diodes. The reactor body consists of two compartments constructed from transparent polymethyl methacrylate sheets. The primary compartment holds the algal culture, which is agitated by means of a recirculating gas-lift. The secondary compartment is used to control the temperature of the system and the wavelength of radiation. The reactor is fitted with probe sensors that monitor the pH, dissolved oxygen, temperature and optical thickness of the algal culture. A membrane-inlet mass spectrometry system has been developed and incorporated into the reactor for dissolved hydrogen measurement and collection. The reactor is hydrogen-tight, modular and fully autoclaveable.     

Metabolic flux analysis of the hydrogen production potential in Synechocystis sp. PCC6803

Hydrogen is a promising energy vector; however, finding methods to produce it from renewable sources is essential to allow its wide-scale use. In that line, biological hydrogen production, although it is considered as a possible alternative, requires substantial improvements to overcome its present low yields. In that direction, genetic manipulation probably will play a central role and from that point of view metabolic flux analysis (MFA) constitutes an important tool to guide a priori most suitable genetic modifications oriented to a hydrogen yield increase.In this work MFA has been applied to analyze hydrogen photoproduction of Synechocystis sp. PCC6803. Flux analysis was carried out based on literature data and several basic fluxes were estimated in different growing conditions of the system. From this analysis, an upper limit for hydrogen photoproduction has been determined indicating a wide margin for improvement. MFA was also used to find a feasible operating space for hydrogen production, which avoids oxygen inhibition, one of the most important limitations to make hydrogen production cost effective. In addition, a set of biotechnological strategies are proposed that would be consistent with the performed mathematical analysis.     

Enhancement of biological hydrogen production using green alga Chlorococcum minutum

It is estimated that the fossil fuel reserves are going to deplete continuously due to extensive usage. In order to cope with this crisis, it is necessary to increase the efforts towards production of biofuels such as biological hydrogen (H₂). It is well-known fact that the biological hydrogen is a clean and ideal energy and liberates high amount of energy per unit mass. Several groups are working for the large scale production of H₂ chemically and also using photosynthetic organisms, but output is not satisfactory. The best way to achieve enhancement of H₂ is through altering the photosynthetic process by applying various stress conditions or by natural selection. In the process of selection, Chlorococcum minutum was found with improved H₂ output when compared to model green alga Chlamydomonas reinhardtii in a massively parallel and competitive high-throughput screen of different green algae. Both the species belongs to class chlorophyceae of green algae and live in fresh water conditions. In extent various light, pH and temperature conditions were applied and achieved the enhancement of H₂ production in this species under in vitro settings. Augmented hydrogenase activity was found in Chlorococcum minutum when compared to model alga and this may be one of the reason behind improved H₂ output. Hence this species may be considered as one of the best species with respect to H₂ production and also this work may be useful for future renewable energy research.     

Fermentative hydrogen production by the newly isolated Enterobacter asburiae SNU-1

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.     

Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii

Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 μM for the hydrogen production. By the re-addition of 30 μM of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H₂ l⁻¹ culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H₂ l⁻¹ culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.     

A novel nutrient control method to deprive green algae of sulphur and initiate spontaneous hydrogen production

The green alga Chlamydomonas reinhardtii has the ability to produce clean and renewable molecular hydrogen through the biophotolysis of water. Hydrogen production takes place under anaerobic conditions, which may be imposed metabolically by depriving the algae of sulphur. Sulphur-deprivation typically requires the spatial and temporal separation of the algal growth and hydrogen production stages. This would typically require separate photobioreactors for each stage as well as a costly and energy intensive medium exchange technique such as centrifugation, making the process difficult to scale up.     

Parameters affecting the growth and hydrogen production of the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has the ability to photosynthetically produce molecular hydrogen (H₂) under anaerobic conditions. It offers a biological route to renewable H₂ production from sunlight and water. Algal growth and H₂ production kinetics must be understood in order to determine appropriate system parameters and develop photobioreactors. Algal biomass should be grown efficiently and economically to attain the high cell densities necessary for H₂ production. The nutrient requirements and process conditions that encourage the growth of dense and healthy algal cultures were explored. Anaerobic conditions were imposed by sulphur deprivation, which requires an exchange of the algal growth medium by centrifugation or dilution. A tubular flow photobioreactor featuring a large surface-to-volume ratio was used to monitor and control the key parameters in the H₂ production process, including pH, dissolved oxygen, optical density, temperature, agitation and light intensity. A cumulative H₂ yield of 3.1 ± 0.3 ml/l of culture was measured.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

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.     

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.     

Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6

Consolidated bioprocessing (CBP) is a promising approach for hydrogen production from lignocellulose owing to its lower cost and higher efficiency. In this study, the newly isolated theromphilic Thermoanaerobacterium sp. strain F6 exhibited the capability of direct utilization of various hemicellulosic and cellulosic materials for hydrogen production, including xylan, Avicel and filter paper etc. Especially, the maximum cumulative hydrogen production reached 370.7 mmoL/L from 60 g/L of xylan. In addition, natural lignocellulosic materials, such as corn cob and sugarcane bagasse without any hydrolytic pretreatment could also be directly utilized as the sole carbon source for hydrogen production. 1822.6 and 826.3 mL H₂/L of hydrogen were produced from corn cob and sugarcane bagasse, respectively. The high hydrogen production from cellulosic and hemicellulosic materials were both benefit from its efficient secretion of hydrolytic enzymes. Thus, Thermoanaerobacterium sp. strain F6 is a potential candidate for effective conversion of lignocellulose to hydrogen through CBP.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

Highly efficient CuO incorporated TiO2 nanotube photocatalyst for hydrogen production from water

Highly dispersed CuO was introduced into TiO₂ nanotube (TNT) made by hydrothermal method via adsorption-calcination process or wet impregnation process, to fabricate CuO incorporated TNT photocatalysts (CuO-TNT) for hydrogen production. It was found that CuO-TNT possessed excellent hydrogen generation activity, which was constantly vigorous throughout 5 h reaction. Depending on the preparation method, hydrogen evolution rates over CuO-TNT were founded in the range of 64.2-71.6 mmol h⁻¹ g⁻¹_catalyst, which was much higher than the benchmark P25 based photocatalysts, and even superior to some Pt/Ni incorporated TNT. This high photocatalytic activity of CuO-TNT was mainly attributed to the unique 1-D tubular structure, large BET surface area and high dispersion of copper component. Compared to wet impregnation, adsorption-calcination process was superior to produce active photocatalyst, since it was prone to produce photocatalyst with more highly dispersed CuO.     

Improvement of hydrogen production with expression of lba gene in chloroplast of Chlamydomonas reinhardtii

An ORF cDNA fragment of one of leghemoglobin genes, lba was cloned from Glycine max and transferred into chloroplasts of Chlamydomonas reinhardtii. More rapidly O₂ consumption, lower O₂ content and higher H₂ output were monitored in the transgenic algal cultures than those in WT cultures either in S-free or S-containing medium. Maximum expression of lba in the transgenic algae consisted with the time when minimal O₂ contents and maximal H₂ evolution occurred. The highest H₂ production achieved in sulfur-free medium for both algal cultures. When restoring sulfate in the medium, H₂ production in the transgenic algal cultures kept steadily around 130–145 μl per bottle while that in WT cultures decreased gradually from 98 μl per bottle at 12.5 μM sulfate to 40 μl per bottle at 100 μM sulfate. The results indicated that heteroexpression of lehemoglaobin genes in chloroplasts of green algae improved H₂ yield by decreasing O₂ content in the medium. This protein had potential to be used in improvement of H₂ production in green algae.     

Improvement of hydrogen production of Chlamydomonas reinhardtii by co-cultivation with isolated bacteria

Three bacteria, named L2, L3 and L4, were isolated from contaminated cultures of Chlamydomonas reinhardtii strain cc849 in laboratory. The phylogenetic analysis based on 16S rDNA sequences showed that L2, L3 and L4 belonged to genus Stenotrophomonas, Microbacterium and Pseudomonas, respectively. The co-cultivation of isolated L2, L3 and L4 with purified algae, respectively, demonstrated that moderate bacterial concentration did not affect algal growth significantly but improved algal H₂ production obviously. The maximal H₂ yields were gained by the co-culture of algae with L2 or L4, about 4.0 times higher than that of the single algal culture. Increased respiration rate or O₂ consumption was the main reason for the enhancement of H₂ yield of the co-cultures.     

New efficient hydrogen process production from organosilane hydrogen carriers derivatives

While the source of hydrogen constitutes a significant scientific challenge, addressing issues of hydrogen storage, transport, and delivery is equally important. None of the current hydrogen storage options, liquefied or high pressure H₂ gas, metal hydrides, etc… satisfy criteria of size, costs, kinetics, and safety for use in transportation. In this context, we have discovered a methodology for the production of hydrogen on demand, in high yield, under kinetic control, from organosilane hydrogen carriers derivatives and methanol as co-reagent under mild conditions catalyzed by a cheap ammonium fluoride salt. Finally, the silicon by-products can be efficiently recycle leading to an environmentally friendly source of energy.     

Process simulation and optimization for enhanced biophotolytic hydrogen production from green algae using the sulfur deprivation method

This article explores the modeling, simulation and optimization of a biophotolytic cyclic process for enhanced hydrogen production from microalgae, employing the sulfur deprivation method. To achieve sulfur deprivation, each process cycle contained two temporally separated steps of sulfur-controlled algae growth and sulfur-deprived anaerobic hydrogen production.Reaction kinetics were modeled via an empirical logistic model. Reaction times, sulfate concentrations, and medium pH levels of each cycle were controlled to optimize the rate and yield of hydrogen production. Consequently, 65% and 23% improved values were obtained, respectively, with a smaller total process time (?11%), higher ratio of algae growth-to-hydrogen production time (29% vs. 21%), buffered pH (7.8), controlled sulfate injection and intermediary algae concentrations. Two- and 15-times higher hydrogen yields were obtained for 2- and 12-times lower initial algae concentrations. The proposed method is a significant tool for the design and optimization of a process for enhanced hydrogen production from microalgae.