Photofermentative hydrogen production using dark fermentation effluent of sugar beet thick juice in outdoor conditions

In the present study, photofermentative hydrogen production on thermophilic dark fermentation effluent (DFE) of sugar beet thick juice was investigated in a solar fed-batch panel photobioreactor (PBR) using Rhodobacter capsulatus YO3 (hup⁻) during summer 2009 in Ankara, Turkey. The DFE was obtained by continuous dark fermentation of sugar beet thick juice by extreme thermophile Caldicellulosiruptor saccharolyticus and it contains acetate (125 mM) and NH₄⁺ (7.7 mM) as the main carbon and nitrogen sources, respectively. The photofermentation process was done in a 4 L plexiglas panel PBR which was daily fed at a rate of 10% of the PBR volume. The DFE was diluted 3 times to adjust the acetate concentration to approximately 40 mM and supplemented with potassium phosphate buffer, Fe and Mo. In order to control the temperature, cooling was provided by recirculating chilled water through a tubing inside the reactor. Hydrogen productivity of 1.12 mmol/L_c/h and molar yield of 77% of theoretical maximum over consumed substrate were attained over 15 days of operation. The results indicated that Rb. capsulatus YO3 could effectively utilize the DFE of sugar beet thick juice for growth and hydrogen production, therefore facilitating the integration of the dark and photo-fermentation processes for sustainable biohydrogen production.     

Photofermentative hydrogen production from volatile fatty acids present in dark fermentation effluents

In the present study, the growth and hydrogen production of Rhodobacter sphaeroides O.U. 001, was investigated in media containing five different volatile fatty acids (VFA) individually (malate, acetate, propionate, butyrate and lactate) and in media containing mixtures of these acids that reflect the composition of dark fermentation effluents. The highest hydrogen production rate was obtained in malate (24 ml_hydrogen/l_reactor h) and the highest biomass concentration was obtained in acetate containing media (1.65 g/l). The substrate conversion efficiencies for different volatile fatty acids were found to vary between 14 and 50%. The malate and butyrate consumption rates were first order with consumption rate constants of 0.026 h⁻¹ and 0.015 h⁻¹, respectively. In the case of substrate mixtures, it was observed that the bacteria consumed acetate first, followed by propionate and then butyrate. It was also found that the consumption rate of the main substrate significantly increased when the minor substrates were depleted.     

Significance of carbon to nitrogen ratio on the long-term stability of continuous photofermentative hydrogen production

The stable and optimized operation of photobioreactors (PBRs) is the most challenging task in photofermentative biological hydrogen production. The carbon to nitrogen ratio (C/N) in the feed is a critical parameter that significantly influences microbial growth and hydrogen production. In this study, the effects of changing the C/N ratio to achieve stable biomass and continuous hydrogen production using fed-batch cultures of Rhodobacter capsulatus YO3 (uptake hydrogenase deleted, hup-) were investigated. The experiments were carried out in 8 L panel PBRs operated in indoor conditions under continuous illumination and controlled temperature. Culture media containing different acetate (40-80 mM) and glutamate (2-4 mM) concentrations were used to study the effects of changing the C/N ratio on biomass growth and hydrogen production. Stable biomass concentration of 0.40 g dry cell weight per liter culture (gDCW/L_c) and maximum hydrogen productivity of 0.66 mmol hydrogen per liter culture per hour (mmol/L_c/h) were achieved during fed-batch operation with media containing 40 mM acetate and 4 mM glutamate, C/N = 25, for a period of over 20 days. A study on the effect of biomass recycling on biomass growth and hydrogen production showed that the feedback of cells into the photobioreactor improved biomass stability during the fed-batch operation but decreased hydrogen productivity.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

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.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

Pilot-scale outdoor photofermentative hydrogen production from molasses using pH control

The duration of photofermentative hydrogen production from sugar-based nutrients is limited by gradual acidification caused by the production of organic acids, leading to suboptimal pH. To address this issue, a custom pH control system was built and installed on a 20 L tubular photobioreactor, and operated under outdoor conditions. Long-term, single-stage hydrogen production from molasses was achieved using the purple non-sulfur bacterium, Rhodobacter capsulatus. The run lasted for 48 days, the longest duration achieved in a tubular photobioreactor on molasses as the only feed. pH was maintained close to its optimum value. High-purity hydrogen (above 90% by mole, on average) and near-complete conversion of sucrose was observed. The highest hydrogen productivity was 0.69 molH₂/(m³.h). On the other hand, hydrogen production was observed to cease after periods of activity. Production resumed after dilution followed by artificial illumination, indicating that the production activity could be recovered during prolonged runs.     

Hydrogen productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate

Hydrogen productivities of different photosynthetic bacteria have been searched on real thermophilic dark fermentation effluents (DFE). The results obtained with potato steam peels hydrolysate (PSP) DFE were compared to glucose DFE. Photobiological hydrogen production has been carried out in indoor, batch photobioreactors using several strains of purple non-sulfur (PNS) bacteria such as Rhodobacter capsulatus (DSM1710), Rhodobacter capsulatus hup- (YO3), Rhodobacter sphaeroides O.U.001 (DSM5864), Rb. sphaeroides O.U.001 hup- and Rhodopseudomonas palustris.The efficiency of photofermentation depends highly on the composition of the effluent and the PNS bacterial strain used. Rb. sphaeroides produced the highest amount of hydrogen on glucose DFE. Rb. capsulatus gave better results on PSP DFE. This study demonstrates that photobiological hydrogen production with high efficiency and productivity is possible on thermophilic dark fermentation effluents. Consequently, a sequential operation of dark fermentation and photofermentation is a promising route to produce hydrogen, and it provides a higher hydrogen yield compared to single step processes.     

Kinetic analysis of photosynthetic growth,hydrogen production and dual substrate utilization by Rhodobacter capsulatus

Rhodobacter capsulatus is purple non-sulfur (PNS) bacterium which can produce hydrogen and CO₂ by utilizing volatile organic acids in presence of light under anaerobic conditions. Photofermentation by PNS bacteria is strongly affected by temperature and light intensity. In the present study we present the kinetic analysis of growth, hydrogen production, and dual consumption of acetic acid and lactic acid at different temperatures (20, 30 and 38 °C) and light intensities (1500, 2000, 3000, 4000 and 5000 lux). The cell growth data fitted well to the logistic model and the cumulative hydrogen production data fitted well to the Modified Gompertz Model. The model parameters were affected by temperature and light intensity. Lactic acid was found to be consumed by first order kinetics. Rate of consumption of acetic acid was zero order until most of the lactic acid was consumed, and then it shifted to first order. The results revealed that the optimum light intensities for maximum hydrogen production were 5000 lux for 20 °C and 3000 lux for 30 °C and 38 °C.     

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.     

Sequential dark and photo-fermentative hydrogen gas production from agar embedded molasses

In this study, molasses and dark fermentation effluent were solidified using agar and used for H₂ production by dark and photo-fermentation. During dark fermentation, the solid jelly form of molasses enabled a slow release of the substrate to the liquid broth hindering fast pH decreases. The initial total sugar concentration, H₂ yield, H₂ rate and lag phase in dark fermentation were 36.2 g/L, 226.24 mL H₂/g TS, 29.85 mL H₂/h and 4.37 h, respectively. Photo-fermentation of 5.77 g TVFA/L embedded dark fermentation effluent did not lead to efficient H₂ production. The best performance in photo-fermentation was obtained with 1.55 g TVFA/L containing diluted dark fermentation effluent. The H₂ yield, H₂ rate and lag phase in photo-fermentation were 870.26 mL H₂/g TVFA, 0.913 mL H₂/h and 54.07 h, respectively. Embedding concentrated substrate using agar can enhance H₂ production performance but only if the release of the substrate does not exceed inhibitory levels and if the rate of diffusion is tolerable for microbial activity.     

Photofermentative hydrogen production from molasses: Scale-up and outdoor operation at low carbon-to-nitrogen ratio

Photofermentative hydrogen production was carried out under outdoor conditions with a Rhodobacter capsulatus strain on molasses, a renewable and sustainable feedstock. An existing photobioreactor design was scaled-up from 9 L to 20 L. The decreased carbon-to-nitrogen (C/N) ratio of 13.0, compared to our previous work, accelerated growth and resulted in a reduced lag period for hydrogen production as well as higher productivities in the exponential phase. However, the low C/N ratio also promoted a high optical density due to growth, limiting light transmission. Still, the maximum productivity was found as 0.47 mol H₂/(m³·h), significantly higher than our result with the smaller reactor volume. High rates of production could not be maintained presumably due to the combined effects of cloudy periods, the aforementioned C/N ratio and decreasing pH. These results suggest that the scale-up was successful and there is potential for further improvement using optimal C/N ratio and cell concentration values.     

Effect of iron and molybdenum addition on photofermentative hydrogen production from olive mill wastewater

Photofermentative hydrogen production from olive mill wastewater (OMW) by Rhodobacter sphaeroides O.U.001 was assessed under iron and molybdenum supplementation. Control cultures were only grown with 2% OMW containing media. The analysis included measurements of biomass accumulation, hydrogen production, pH variations of the medium, and changes in the chemical oxygen demand (COD) of the wastewater. Growth under control and Mo-supplemented experiments yielded about the same amount of biomass (∼0.4 g dry cell weight per L culture). On the other hand, Mo addition slightly enhanced the total volume of H₂ gas production (62 mL H₂), in comparison with the control reactor (40 mL H₂). Fe-supplemented cultures showed a significant increase on H₂ production (125 mL H₂), tough having a longer lag time for the observation of the first H₂ bubbles (24 h), compared to the control (15 h) and Mo-supplemented ones (15 h). Fe-added cultures also yielded better wastewater treatment by achieving 48.1% degradation of the initial chemical oxygen demand (COD) value compared to the control reactor having 30.2% COD removal efficiency. Advances described in this work have the potential to find applications in hydrogen industry while attempting an effective management of cheap feedstock utilization.     

Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus

The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In the present study, the relationship between light intensity and hydrogen production has been modelled in order to predict both the rate of hydrogen production and the amount of hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using lactate as carbon and hydrogen source. The rate of hydrogen production increased with increasing light intensity in a manner that was described by a static Baly model, modified to include the square of the light intensity. In agreement with previous studies, the kinetics of substrate utilization and growth of R. capsulatus was represented by the classical Monod or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen.     

Prevention of substrate and product inhibitions by using a dilution strategy during dark fermentative hydrogen production from molasses

Substrate and product inhibitions have a significant effect on dark fermentative hydrogen gas production. Particularly, rapid formation of volatile fatty acids leads to fast pH decreases shifting the metabolic pathway. Therefore, controlling volatile fatty acid accumulation has great importance in maintaining effective hydrogen production. In this context, a dilution strategy was applied to regulate volatile fatty acids levels within the desired concentration range. A three-factor Box-Behnken statistical experiment design was established to assess the effects of dilution time, dilution percentage and initial COD concentration on hydrogen formation yield and rate. Highest hydrogen yield (7.7 mL H₂/mL_reactor) and rate (21. 47 mL H₂/h or 9.38 mmol/L_reactor.h) were achieved when 85 gCOD/L containing fermentation media was diluted with a percentage of 130 of the initial working volume at the 3rd hour of the fermentation period. Moreover, this strategy enabled to start fermentation with 55 g glucose/L.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

Optimization of photosynthetic hydrogen production from acetate by Rhodobacter sphaeroides RV

In this study, hydrogen production by Rhodobacter sphaeroides RV from acetate was investigated. Ammonium sulphate and sodium glutamate were used to study the effects of nitrogen sources on photosynthetic hydrogen production. The results showed the optimal concentrations for ammonium sulphate and sodium glutamate were in the range of 0.4–0.8 g/L. Orthogonal array design was applied to optimize the hydrogen-producing conditions of the concentrations of yeast, FeSO₄ and NiCl₂. The theoretical optimal condition for hydrogen production was as follow: yeast 0.1 g/L, FeSO₄ 100 mg/L and NiCl₂ 20 mg/L.