Scale-up studies for stable,long-term indoor and outdoor production of hydrogen by immobilized Rhodobacter capsulatus

Photofermentative hydrogen production by immobilized Rhodobacter capsulatus YO3 was carried out in a novel photobioreactor in sequential batch mode under indoor and outdoor conditions. Long-term H₂ production was realized in a 1.4 L photobioreactor for 64 days using Rhodobacter capsulatus YO3 immobilized with 4% (w/v) agar on 5 mM sucrose and 4 mM glutamate. The highest hydrogen yield (19 mol H₂/mol sucrose) and hydrogen productivity (0.73 mmol H₂ L^?1 h^?1) were achieved indoors on 5 mM sucrose. The effect of initial sucrose concentration (5 mM, 10 mM, and 20 mM) on hydrogen production was also investigated. Sustained hydrogen production was carried out under natural, outdoor conditions as well. For the outdoor experiments, the highest hydrogen productivity and yield were obtained as 0.87 ± 0.06 mmol H₂ L^?1 h^?1 and 6.1 ± 0.2 mol H₂/mol sucrose, respectively on 10 mM sucrose. Furthermore, this system prevented sudden pH drops and fluctuations caused by the utilization of sucrose throughout the process. These results demonstrate that a proper immobilization setup can lead to long-term efficient and robust hydrogen production even under naturally varying conditions.     

The exploration of hydrogen production promoting mechanism of Rhodobacter sphaeroides by expressing tetA under tetracycline stress

Photo-fermentative hydrogen production rate improving has been a key limiting link in the field of biological hydrogen production by photosynthetic bacterium (PSB) in recent years. In our previous study, apparent hydrogen production enhancement was observed in R. sphaeroides HY01 with tetA expressed under tetracycline (Tc), which attracted our interests. It was found that Tc-resistant strains always presented stable hydrogen production promotion on the maximum hydrogen production rate (R_m), which was approximately 30% with Tc addition. Figuring out the underlying connections between Tc resistance and hydrogen production of R. sphaeroides may provide new methods for obtaining engineered PSB with high hydrogen production performance. In this study, basing on experimental tests, hydrogen promotion of resistant strain was found to be Tc dose-dependent. Some relevant gene expression differences on hydrogen production of Rodobacter sphaeroides induced by the expression of TetA and the addition of Tc were also tested and discussed.     

Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H₂ production after pretreatment. However, the pretreatment methods have different influence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/cellusase. The hydrolysis rates increased from 45.51% by ddH₂O-treatment to 60.79% by diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H₂ production vary among different hydrolysates. Photo-fermentative H₂ production of strain R. capsulatus JL1 and mutant JL1601 (cheR2^-) with hydrolysates were investigated. The maximum H₂ yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H₂ yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2^-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2^-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH₂O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H₂ production for mutant JL1601 (cheR2^-) but not for strain JL1.     

High NH3N tolerance of a cheR2-deletion Rhodobacter capsulatus mutant for photo-fermentative hydrogen production using cornstalk

A mutant, JL1601, with a cheR2 gene deletion was constructed with Rhodobacter capsulatus JL1 as the wild type strain. The effects of pH, ammonia nitrogen concentration and light intensity on the photo-fermentative hydrogen production performance of JL1601 were studied. Suitable pH range for hydrogen production with the cheR2^- mutant migrated from faintly acid to weak alkaline condition, and R.capsulatus JL1601 (cheR2^-) showed higher NH₃N tolerance. The mutant performed better H₂ production performance than the wildtype under the same illumination condition. The maximum H₂ yield and rate of R.capsulatus JL1601 (cheR2^-) obtained to be 4406.7 ± 45.9 mL/L and 54.1 ± 1.6 mL/(L·h) with the mixture of acetate and butyrate as the carbon sources, which were increased by 36.3% and 12.3% in comparison to those of wildtype, respectively. Practical experiments using acid-enzymatic pretreated cornstalk as the carbon source indicated that, the maximum hydrogen yields by the mutant JL1601 (cheR2^-) was up to 224.85 ± 5.18 mL-H₂/g-cornstalk, which increased by 171.43% compared with that of wild type strain.     

Value analysis for commercialization of fermentative hydrogen production from biomass

In addition to producing hydrogen gas, biohydrogen production is also used to process wastewater. Therefore, this study specifically conducted value analyses of two different scenarios of fermentative hydrogen production from a biomass system: to increase the value of a wastewater treatment system and to specifically carry out hydrogen production. The analytical results showed that fermentative hydrogen production from a biomass system would increase the value of a wastewater treatment system and make its commercialization more feasible. In contrast, fermentative hydrogen production from a biomass system designed specifically for producing hydrogen gas would have a lower system value, which indicated that it is not yet ready for commercialization. The main obstacle to be overcome in promoting biohydrogen production technology and system application is the lack of sales channels for the system's products such as hydrogen gas and electricity. Thus, in order to realize its commercialization, this paper suggests that governments provide investment subsidies for the use of biohydrogen production technology and establish a buy-back tariff system for fuel cells.     

“Effect of light intensity and the light: dark cycles on the long term hydrogen production of Chlamydomonas reinhardtii by batch cultures”

The photomixotrophic hydrogen production was investigated in sulfur deprived Chlamydomonas reinhardtii cultures. The cultures were exposed to continuous illumination of various light intensities in 27-day batches. Light intensity of 70 × 2 ??E m⁻² s⁻¹ was selected for hydrogen production. Subsequent experiments involving 27-day long light:dark cycles were conducted at the selected light intensity. The cycles consisted of hour divisions (h:h; 18:6, 14:10, 12:12) or minute divisions (min:min; 45:15, 35:25, 30:30). The results showed an adverse effect of the light:dark cycles on hydrogen production. All experiments, irrespective of the type of illumination indicated that cultures needed a lag phase for production and the highest hydrogen production was obtained during first 7-10 days of production reaching a peak in the first 5 days.     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

Effect of the nitrogen source on the hydrogen production metabolism and hydrogenases of Clostridium butyricum CWBI1009

Investigations were carried out to determine the effect of various concentrations of organic and ammonium nitrogen sources on fermentative hydrogen production by the strain Clostridium butyricum CWBI1009. The results indicated that the H₂-producing metabolism of the strain is favoured within the range (0.56–0.062 g_N/L) of peptone and (NH₄)₂SO₄. Optimal overall performance (i.e. 1.43 ± 0.08 mol H₂/mol glucose and 1.08 ± 0.03 mL_H2/h, respectively) was achieved with 0.062 g_N/L of casein peptone. The study of the amino acid uptake and the gene expression pattern for four [FeFe]-hydrogenases and the nitrogenase showed that nitrogen was in excess in all the experiments with a nitrogen concentration above 0.062 g_N/L and, at that optimal concentration, the expression of the HydB2 gene would be responsible for the much higher H₂ yield.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of ground wheat solution: Effects of light source and light intensity

Dark fermentation effluent of wheat powder solution was subjected to light fermentation for bio-hydrogen production using different light sources and intensities. Tungsten, fluorescent, infrared (IR), halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Pure culture of Rhodobacter sphaeroides-RV was used in batch light fermentation experiments. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (CHF, 252 ml) and yield (781 ml H₂ g⁻¹ TVFA). In the second set of experiments, light fermentations were performed at different light intensities (1–10 klux) using halogen lamp. The optimum light intensity was found to be 5 klux (approx. 176 Wm⁻²) resulting in the highest CHF (88 ml) and hydrogen yield (1037 ml H₂ g⁻¹TVFA). Hydrogen formation was limited by the availability of light at low light intensities below 5 klux and was inhibited by the excess light above 5 klux.     

Effect of culture conditions on the kinetics of hydrogen production by photosynthetic bacteria in batch culture

Biochemical kinetic characteristics of photo-fermentative hydrogen production were experimentally and numerically investigated to optimize the photo-fermentation hydrogen-producing process in this work. It is found that a maximum specific growth rate of 0.26 h⁻¹ was achieved under the optimal conditions of illumination intensity 6000 lux, 30 °C culture temperature and pH 7.0 of culture medium. These experimental results also led to an empirical formula of the maximum specific microbial growth rate (μ_max) as a function of illumination intensity, pH and temperature. With the empirical formula, the modified Monod equation along with the kinetic equations for biomass growth, glucose consumption and hydrogen production is then developed to simulate the photofermentation hydrogen-producing process. The modeling results are in good agreements with the experimental data, indicating that the developed kinetic models are able to objectively describe the characteristics of hydrogen production by PSB under different culture conditions.     

Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710

Photofermentative hydrogen production is influenced by several parameters, including feed composition, pH levels, temperature and light intensity. In this study, experimental results obtained from batch cultures of Rhodobacter capsulatus DSM 1710 were analyzed to locate the maximum levels for the rate and yield of hydrogen production with respect to temperature and light intensity. For this purpose, a 3^k general full factorial design was employed, using temperatures of 20, 30 and 38 °C and light intensities of 100, 200 and 340 W/m². ANOVA results confirmed that these two parameters significantly affect hydrogen production. Surface and contour plots of the regression models revealed a maximum hydrogen production rate of 0.566 mmol H₂/L/h at 27.5 °C and 287 W/m² and a maximum hydrogen yield of 0.326 mol H₂/mol substrate at 26.8 °C and 285 W/m². Validation experiments at the calculated optima supported these findings.     

The feasibility of heating on tumor periphery by using high intensity focused ultrasound thermal surgery

The aim of this study is to investigate the feasibility of the heating on the tumor periphery by using high intensity focused ultrasound (HIFU) during thermal surgery. The pressure field induced by high intensity focused ultrasound transducer was solved by the Rayleigh–Sommerfeld diffraction integral. The temperature distribution was solved by the Pennes bioheat transfer equation. Numerical results show that even in the tumor periphery with high blood perfusion the thermal lesion can effectively cover the wanted therapeutic region for a rapid HIFU heating. The modality of heating on the tumor periphery by using high intensity focused ultrasound may provide an approach to eradication of a solid tumor with high blood perfusion in the periphery.     

Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented.Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design.Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen.Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB.All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.     

Luminous intensity solid of tubular light guide and its characterization using “asymmetry parameter”

The luminous intensity solid of hollow light guide is determined using an extended HOLIGILM solution. The solution concept enables to simulate luminous intensity solids for various light guides and sky luminance patterns. Angular distribution of light emitted from bottom interface of the light guide strongly depends on how the illumination system is designed. Any beam undergoes many reflection events which influence significantly its direction below the optical interface. As a consequence, the computed luminous intensity solid may show many peak during sunny days. After interaction with tube the light beams suffer from imperfect reflections resulting in non-uniform luminous intensity solid even if the sky luminance is constant. The directionality of the light below the pipe is characterized by asymmetry parameter which is a cosine-weighted integral of luminous intensity. Such a single-value can serve as supplementary parameter that quantifies a measure of downward luminous emissions.     

Genetically engineered deletion in the N-terminal region of nifA1 in R. capsulatus to enhance hydrogen production

NifA is the primary activator of nitrogenase, and the N-terminal domain of nifA is sensitive to ammonium concentration. In this work, a mutant Rhodobacter capsulatus ZX01 with a genetically engineered deletion in the N-terminal region of nifA1 was constructed by employing overlap extension PCR to mitigate the inhibition of ammonium on nitrogenase expression in photosynthetic bacteria. The effects of different ammonium ion concentrations on the growth and photo-fermentative hydrogen production performance of wild-type strain R. capsulatus SB1003 and mutant ZX01 with glucose and volatile fatty acids as the carbon sources were studied, respectively. When the ratio of NH₄⁺-N was 20% and 30%, the hydrogen yield of the mutant ZX01 was enhanced by 14.8% and 20.9% compared with that of R. capsulatus SB1003 using 25 mM acetic acid and 34 mM butyric acid as the carbon source, respectively. In comparison, using 30 mM glucose as the carbon source, the hydrogen yield of ZX01 was increased by 17.7% and 22.2% compared with that of R. capsulatus SB1003 when the ratio of NH₄⁺-N was 20% and 30%, and the nitrogenase activity of ZX01 was also enhanced by 38.0% and 47.6%, respectively. When using 10 mM NH₄⁺ as a single nitrogen source, ZX01 showed a 2.6-fold increase in H₂ production. These results indicated that ZX01 demonstrated higher ammonium tolerance and better hydrogen production performance than the wild-type. The deletion in the N-terminal region of nifA1 could partially de-repress the nitrogenase activity inhibited by ammonium.