Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control

The present study investigated the production of hydrogen (H₂) and ethanol from glucose in an Anaerobic Continuous Stirred Tank Reactor (ACSTR). Effects of hydraulic retention time (HRT) and pH on the preference of producing H₂ and/or ethanol and other soluble metabolic products in an open anaerobic enriched culture were studied. Production rates of H₂ and ethanol increased with the increase of biomass concentration. Open anaerobic fermentation was directed and managed through on-line pH control for the production of H₂ or ethanol. Hydrogen was produced by ethanol and acetate-butyrate type fermentations. pH has strong effect on the H₂ or ethanol production by changing fermentation pathways. ACSTR produced mainly ethanol at over pH 5.5 whereas highest H₂ production was obtained at pH 5.0. pH 4.9 favored the lactate production and accumulation of lactate inhibited the biomass concentration in the reactor and the production of H₂ and ethanol. The microbial community structure quickly responded to pH changes and the Clostridia dominated in ACSTR during the study. H₂ production was maintained mainly by Clostridium butyricum whereas in the presence of Bacillus coagulans glucose oxidation was directed to lactate production.     

Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus

Stripping gas is generally used to improve hydrogen yields in fermentations. Since CO₂ is relatively easy to separate from hydrogen it could be an interesting stripping gas. However, a higher partial CO₂ pressure is accompanied with an increased CO₂ uptake in the liquid, where it hydrolyses and induces an increased requirement of NaOH to maintain the pH. This enhances the osmotic pressure in the culture by 30%, which inhibited the growth of Caldicellulosiruptor saccharolyticus. Indications for this conclusion are: i) Inhibition could almost completely be circumvented by reducing the bicarbonate through decreasing the pH (from 6.5 to 5.5), ii) Growth rates were reduced by 60 ± 10% at an osmotic pressure of 0.218 ± 0.005 osm/kg H₂O independently of the stripping gas, iii) Increased extracellular DNA and protein concentrations were observed as a function of the osmotic pressure. In addition to growth inhibition, the increased sodium bicarbonate in the effluent will contribute to a negative environmental impact when applied at industrial scale.     

Escherichia coli wild type and hydrogenase mutant cells growth and hydrogen production upon xylose and glycerol co-fermentation in media with different buffer capacities

In this study, the new strategy for long term bio-hydrogen (H₂) production using different substrates and waste materials is presented. Growth characteristics and H₂ production were investigated upon consumption of 0.4% xylose and 1% glycerol alone (which were optimal) or their mixture by Escherichia coli BW25113 wild type parental strain (PS) and ΔhyaB, ΔhybC, ΔhycE, ΔhyfG mutants with genes deletions for key subunits of hydrogenase (Hyd)-1 to Hyd-4, respectively, in high and low buffer capacity peptone (HPM, LPM) mediums, pH 5.5 and 7.5. Overall, pH 5.5 negatively affected bacterial growth and H₂ production. At pH 7.5, apart from Hyd-3 and Hyd-4 mutants, upon growth of PS, Hyd-1 and Hyd-2 mutants drop of Pt redox electrode readings from positive (～+150 mV) to negative (of ?400 to ?550 mV) values was detected during log growth phase mentioning H₂ formation. Xylose and glycerol co-utilization did not affect PS and Hyd-1 and Hyd-2 mutant's biomass and H₂ formation during log growth phase in LPM, but ～1.5 fold stimulated these parameters, especially in HPM, pH 7.5, during prolonged 96 h bacterial growth. Roles of Hyd-3 and Hyd-4 in H₂ production; and Hyd-1 and Hyd-2 in H₂ oxidation during bacterial log growth phase were stated under xylose and glycerol co-fermenting conditions. The results obtained might be valuable for industrial long term H₂ production by bacteria using mixture of carbon sources and combining various organic waste materials.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

The influence of hydrogen production on the formation of metabolic pathways and regulation of ΔpH in Escherichia coli

Hydrogen (H₂) metabolism in Escherichia coli occurs via reversible membrane-associated hydrogenase enzymes (Hyd). Hyd-3 and Hyd-4 with formate dehydrogenase H (FDH-H) form formate hydrogen lyase complexes. The changes of metabolic pathways and ΔpH (pH_in-pH_ex) regulation during fermentation of glucose, glycerol and formate in non H₂-producing hypF (lack of all Hyds) and fdhF (lack of FDH-H) mutants at pH 7.5 were investigated. It was shown that specific growth rate was higher by ～23% in hypF and fdhF, compared to wild type (wt), suggesting the negative effect of H₂ on bacterial growth. Moreover, it was shown that H₂ generation did not have a vital role in glucose and glycerol utilization rate at 0–72 h. The utilization of external formate was detected in wt (～2.6 mM) and hypF (～0.68 mM), but not in fdhF, due to the absence of enzyme responsible for formate metabolism. Nevertheless, the changes in ΔpH were not evident at 3 h. The ratio of generated end-products and regulation of ΔpH at late log (6 h) and exponential phase (24, 72 h) were various in hypF and fdhF due to formate disproportionation in hypF and proton generation, therewith absence of H₂ generation. Taken together it can be concluded that bacteria regulate generation of fermentation end-products via balancing the concentration of acids and ethanol to maintain ΔpH and redox potential values. The results obtained are important for development and regulation of H₂ production technology when applying mixed carbon sources.     

Light-driven biological hydrogen production by Escherichia coli mediated by TiO2 nanoparticles

Photocatalytic hydrogen production using an inorganic bio-hybrid system can contribute to the proficient utilization of light energy, but it would necessitate the development of novel approaches for preparing a new hydrogen-producing biocatalyst. In this study, we developed a hybrid system to produce hydrogen, whereby the highly efficient light-harvesting inorganic semiconductor (TiO₂) was mixed with Escherichia coli to form a biocatalyst with the addition of an electron mediator (MV²⁺) under different visible light irradiation. Under this hybrid system, the hydrogen production by E. coli was light intensity-dependent showing maximum production at 2000 W m⁻², with a 2-fold increase in the hydrogen production compared to that without TiO₂. The experiments on the continued cycle of hydrogen production revealed that the production could be continued for at least 3 cycles of 5 h incubation for each. A possible pathway utilizing glucose for hydrogen production by the hybrid system was proposed based on the analysis of the levels of metabolites. A feasibility study was also conducted using natural sunlight for hydrogen production by the hybrid system. Overall results demonstrated that whole cells of E. coli could be employed for photocatalytic hydrogen production where the intactness of the E. coli was retained under experimental conditions.     

Hydrogen production from fossil and renewable sources using an oxygen transport membrane

Oxygen transport membranes (OTMs) made of mixed ion-electron conductors can be used to increase the production of hydrogen from fossil and renewable sources. This study describes two methods for producing hydrogen with La_0.7Sr_0.3Cu_0.2Fe_0.8O_3−δ (LSCF7328), an OTM material that is easily prepared, exhibits good mechanical properties, and is stable in severe gas conditions. In tests with thin-film (thickness ≈22 μm) LSCF7328 membranes, hydrogen was produced by flowing simulated product streams from CO₂ gasification of coal on one side of the OTM and steam on the other side. In this method, the so-called coal gas on the oxygen-permeate side drives the removal of oxygen from the other side of the OTM, where hydrogen and oxygen are produced by water splitting. With CO (99.5% purity) flowing on the oxygen-permeate side, the hydrogen production rate was measured to be ≈4.7 cm³/min-cm² at 900 °C, indicating that hydrogen can be produced at a significant rate by using product streams from coal gasification. This process also yields a CO₂-rich product stream that is ready for sequestration. In another test, a tubular LSCF7328 was found to increase the hydrogen production from ethanol reforming by supplying high-purity oxygen from air.     

Biological hydrogen production by Anabaena sp. - Yield, energy and CO2 analysis including fermentative biomass recovery

This paper presents laboratory results of biological production of hydrogen by photoautrotophic cyanobacterium Anabaena sp. Additional hydrogen production from residual Cyanobacteria fermentation was achieved by Enterobacter aerogenes bacteria. The authors evaluated the yield of H₂ production, the energy consumption and CO₂ emissions and the technological bottlenecks and possible improvements of the whole energy and CO₂ emission chain.The authors did not attempt to extrapolate the results to an industrial scale, but to highlight the processes that need further optimization.The experiments showed that the production of hydrogen from cyanobacteria Anabaena sp. is technically viable. The hydrogen yield for this case was 0.0114 kgH₂/kg_biomass which had a rough energy consumption of 1538 MJ/MJH₂ and produced 114640 gCO₂/MJH₂. The use of phototrophic residual cyanobacteria as a substrate in a dark-fermentation process increased the hydrogen yield by 8.1% but consumed 12.0% more of energy and produced 12.1% more of CO₂ showing that although the process increased the overall efficiency of hydrogen production it was not a viable energy and CO₂ emission solution. To make cyanobacteria-based biofuel production energy and environmentally relevant, efforts should be made to improve the hydrogen yield to values which are more competitive with glucose yields (0.1 kgH₂/kg_biomass). This could be achieved through the use of electricity with at least 80% of renewables and eliminating the unessential processes (e.g. pre-concentration centrifugation).     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

Effect of sulfate on hydrogen production from the organic fraction of municipal solid waste using co-culture of E. coli and Enterobacter aerogenes

In the present study, the effect of sulfate on the hydrogen production from the organic fraction of municipal solid (OFMSW) waste using co-culture of Enterobacter aerogenes and E. coli has been studied under varying pH conditions. The presence of sulfate in the feedstock declines hydrogen production efficiency. To evaluate the effect of sulfate on hydrogen production from OFMSW, COD/sulfate ratio of 17.5, 15.0, 12.5, 10.0, 7.5, 5.0 and 2.5 were applied at different pH conditions (i.e. pH 5.5, 6.0 and 6.5). The hydrogen production continuously declined with the decreasing COD/sulfate ratio and increase in pH. The cumulative hydrogen production decreased from 220.8 ± 10.5 mL in control to a minimum of 98.3 ± 10.5 mL, 74.4 ± 10.4 mL, and 44.6 ± 2.6 mL at pH 5.5, 6.0 and 6.5 respectively. The major content of gaseous composition included hydrogen and CO₂ at higher COD/sulfate ratio and low pH, while H₂S formation started with the decrease in COD/sulfate ratio and increase in the pH. Similarly, sulfate removal efficiency was found to be influenced by COD/sulfate ratio and pH condition. Soluble metabolite analysis revealed that total volatile fatty acid concentration was not affected by sulfate addition. Thus, Sulfate removal is essential prior to fermentation in order to improve hydrogen yield.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.     

Evaluation of hydrogen metabolism by Escherichia coli strains possessing only a single hydrogenase in the genome

The four hydrogenase isozymes; hydrogenase 1 (Hyd-1), hydrogenase 2 (Hyd-2), hydrogenase 3 (Hyd-3) and hydrogenase 4 (Hyd-4) of Escherichia coli have been reported for their crucial functions in the hydrogen metabolism; however, their distinctive roles could not be completely understood. In this study, four ideal hydrogenase operon mutants, Δhyb hyc hyf, Δhya hyc hyf, Δhya hyb hyf, and Δhya hyb hyc, in which only a single hydrogenase is intact in the genome, were constructed as well as one quadruple mutant (Δhya hyb hyc hyf) that all four hydrogenase operons were deleted. First, single operon mutants and single-gene mutants for each hydrogenase showed different hydrogen productivity and growth in the anaerobic fermentation, indicating that bacterial phenotype regarding the hydrogen metabolism via the deletion of each operon is different with that of each single gene. Then, 4 triple hydrogenase operon mutants and one quadruple mutant were investigated to evaluate the hydrogen metabolism (hydrogen production and uptake) using glucose or glycerol as a substrate of hydrogen fermentation. With both the carbon sources, only Hyd-2 and Hyd-3 were able to produce hydrogen. Furthermore, all the hydrogenases showed hydrogen uptake activity. In addition, no hydrogen production and hydrogen uptake were detected in the quadruple mutant which does not have all 4 hydrogenases. Hydrogen production from Hyd-2 and Hyd-3 was further confirmed by complementing their operons in the cloning vector pBR322.     

Optimization of catholyte concentration and anolyte pHs in two chamber microbial electrolysis cells

The hydrogen production rate in a microbial electrolysis cell (MEC) using a non-buffered saline catholyte (NaCl) can be optimized through proper control of the initial anolyte pH and catholyte NaCl concentration. The highest hydrogen yield of 3.3 ± 0.4 mol H₂/mole acetate and gas production rate of 2.2 ± 0.2 m³ H₂/m³/d were achieved here with an initial anolyte pH = 9 and catholyte NaCl concentration of 98 mM. Further increases in the salt concentration substantially reduced the anolyte pH to as low as 4.6, resulting in reduced MEC performance due to pH inhibition of exoelectrogens. Cathodic hydrogen recovery was high (r_cat > 90%) as hydrogen consumption by hydrogenotrophic methanogens was prevented by separating the anode and cathode chambers using a membrane. These results show that the MEC can be optimized for hydrogen production through proper choices in the concentration of a non-buffered saline catholyte and initial anolyte pH in two chamber MECs.     

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.     

Thermodynamic analysis of combined unit of biomass gasifier and tar steam reformer for hydrogen production and tar removal

A combined unit of biomass gasifier and tar steam reformer (CGR) was proposed in this study to achieve simultaneous tar removal and increased hydrogen production. Tar steam reforming calculations based on thermodynamic equilibrium were carried out by using Aspen Plus software. Thermodynamic analysis reveals that when selecting appropriate operating conditions, exothermic heat available from the gasifier could sufficiently supply to the heat-demanding units including feed preheaters, steam generator and reformer. The effects of gasification temperature (T_gs), reforming temperature (T_ref) and steam-to-biomass ratio (S:BM) on percentages of tar removal and improvement of H₂ production were investigated. It was reported that the CGR system can completely remove tar and increase H₂ production (1.6 times) under thermally self-sufficient condition. The increase of H₂ production is mainly via the water–gas shift reaction.     

Hydrogen production upon UV-light irradiation of Cu/TiO2 photocatalyst in the presence of alkanol-amines

Photocatalytic hydrogen production from alkanol-amines as sacrificial agents by using Cu-modified TiO₂–P25 prepared via in situ photodeposition of cupric ions under UV-A light irradiation was investigated. A direct comparison among different sacrificial agents (monoethanolamine, diethanolamine, and triethanolamine) was preliminarily performed. Diethanolamine was selected for further investigation, due to its higher hydrogen production rate with respect to other alkanol-amines. Effect of pH, starting concentration of sacrificial agent, and catalyst/co-catalyst loads were studied. Solution pH exerts major impact on the photoefficiency for hydrogen generation: reaction mechanisms at different pH values were extensively examined. For the first time, a validated kinetic model estimated the unknown rate constants of (i) reaction between diethanolamine and photogenerated holes, (ii) proton reduction, and (iii) diethanolamine adsorption on the photocatalyst surface.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.