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.     

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h^?1 and the average pseudo-steady state hydrogen production rate was 36 mL H₂ L^?1 medium h^?1. The cumulative hydrogen production was 430 mL H₂ L^?1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H₂ L^?1 h^?1 and the cumulative hydrogen production was 517 mL H₂ L^?1 medium. Reducing the feed rate to less than 48 mL h^?1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.     

Selection of metabolic pathways for continuous hydrogen production under thermophilic and mesophilic temperature conditions in anaerobic fluidized bed reactors

This study evaluated the influence of hydraulic retention time (HRT) on hydrogen (H₂) production in anaerobic fluidized bed reactors at mesophilic (30 °C, AFBR-M) and thermophilic (55 °C, AFBR-T) temperatures. Reactors were fed sucrose-based synthetic wastewater (5000 mg chemical oxygen demand·L^?1) in the HRT of 8, 6, 4, 2, or 1 h. H₂ production rate increased from 67.8 ± 14.8 to 194.9 ± 57.0 ml H₂·h^?1 L^?1 (AFBR-T) and from 72.0 ± 10.0 to 344.4 ± 74.0 mL H₂·h^?1·l^?1 (AFBR-M) when HRT decreased from 8 to 1 h. Maximum H₂ yields for AFBR-T and AFBR-M were 1.93 ± 0.21 and 2.68 ± 0.48 mol H₂·mol^?1 sucrose, respectively. The main metabolites were acetic acid (31.3%–41.5%) and butyric acid (10.2%–20.7%) (AFBR-M) and acetate (20.1%–39.3%) and ethanol (14.3%–29.9%) (AFBR-T). Denaturing gradient gel electrophoresis profiles revealed selective enrichment of microbial populations responsible for H₂ production by the aceto-butyric route (AFBR-M) and ethanol-type fermentation (AFBR-T).     

Production of butanol and polyhydroxyalkanoate from industrial waste by Clostridium beijerinckii ASU10

This study demonstrated a biotechnological approach for simultaneous production of low‐cost H₂, liquid biofuels, and polyhydroxyalkanoates (PHAs) by solventogenic bacterium (Clostridium beijerinckii) from renewable industrial wastes such as molasses and crude glycerol. C beijerinckii ASU10 (KF372577) exhibited considerable performance for hydrogen production of 5.1 ± 0.84 and 11 ± 0.44 mL H₂ h^?1 on glycerol and sugarcane molasses, respectively. The total acetone‐butanol‐ethanol (ABE) generation from glycerol and molasses was 9.334 ± 2.98 and 10.831 ± 4.1 g L^?1, respectively. ABE productivity (g L^?1 h^?1) was 0.0486 and 0.0564 with a yield rate (g g^?1) up to 0.508 and 0.493 from glycerol and molasses fermentation, respectively. The PHA yields from glycerol and sugarcane molasses were 84.37% and 37.97% of the dried bacterial biomass, respectively. Additionally, the ultrathin section of C beijerinckii ASU10 showed that PHA granules were accumulated more densely on glycerol than molasses. Gas chromatography–mass spectrometry (GC‐MS) analysis confirmed that the PHAs obtained from molasses fermentation included 3‐hydroxybutyrate (47.3%) and 3‐hydroxyoctanoate (52.7%) as the main constituents. Meanwhile, 3‐hydroxybutyrate represented the sole monomer of PHA produced from glycerol fermentation. This study demonstrated that C beijerinckii ASU10 (KF372577) is a potent strain for low‐cost PHA production depending on its high potential to produce high‐energy biofuel and other valuable compounds from utilization of organic waste materials.     

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.     

Enhancement of fermentative hydrogen production by Thermotoga maritima through hyperthermophilic anaerobic co-digestion of fruit-vegetable and fish wastes

In this work, different proportions of model fruit and vegetable wastes (MFVW) and acid hydrolyzed fish wastes (AHFW) were used for hydrogen production in a minimum culture medium based on seawater. Experiments were performed in pH-controlled Stirred Tank Reactor (STR) with or without the addition of nitrogen and sulfur sources. The total H₂ production and the maximum hydrogen productivity of T. maritima in the culture medium, containing MFVW and AHFW (45 mmol L^?1 carbohydrates) at a C/N ratio of 12, were 132 mmol L^?1 and 15 mmol h^?1 L^?1, respectively. However, tripling the concentration of carbohydrates to reach a C/N ratio of 22, has increased two times the maximum H₂ productivity (28 mmol h^?1 L^?1) due to the improvement in nutrient balance. The cumulative H₂ production was 285 mmol L^?1, yielding a potential energy generation of 0.1210³ MJ ton^?1 wastes, which could be an interesting alternative for energy recovery.     

Continuous hydrogen production from cofermentation of sugarcane vinasse and cheese whey in a thermophilic anaerobic fluidized bed reactor

The co-fermentation of vinasse and cheese whey (CW) was evaluated in this study by using two thermophilic (55° C) anaerobic fluidized bed reactors (AFBRs). In AFBR using vinasse and CW (AFBR-V-CW), the CW was added in increasing proportions (2, 4, 6, 8, and 10 g COD.L^?1) to vinasse (10 g COD.L^?1) to assess the advantage of adding CW to vinasse. By decreasing the hydraulic retention time (HRT) from 8 h to 1 h in AFBR-V, maximum hydrogen yield (HY), production rate (HPR), and H₂ content (H₂%) of 1.01 ± 0.06 mmol H₂.g COD^?1, 2.54 ± 0.39 L H₂.d^?1.L^?1, and 47.3 ± 2.9%, respectively, were observed at an HRT of 6 h. The increase in CW concentration to values over 2 g COD.L^?1 in AFBR-V-CW decreased the HY, PVH, and H₂%, with observed maximum values of 0.82 ± 0.07 mmol H₂.g COD^?1, 1.41 ± 0.24 L H₂.d^?1.L^?1, and 55.5 ± 3.7%, respectively, at an HRT of 8 h. The comparison of AFBR-V-CW and AFBR-V showed that the co-fermentation of vinasse with 2 g COD.L^?1 of CW increased the HPR, H₂%, and HY by 117%, 68%, and 82%, respectively.     

Performance of batch solid state fermentation for hydrogen production using ground wheat residue

Ground wheat (21 g) was subjected to batch solid state dark fermentation for bio-hydrogen production. Clostridium acetobutylicum (B-527) was used as the culture of dark fermentation bacteria at mesophilic conditions. Effects of moisture content on the rate and yield of bio-hydrogen formation were investigated. The highest CHF (1222 ml), hydrogen yield (63 ml H₂ g^?1 starch), formation rate (10.64 ml H₂ g^?1 starch h^?1) and specific hydrogen formation rate (0.28 ml H₂ g^?1 biomass h^?1) were obtained with a moisture content of 80%. Nearly complete starch hydrolysis and glucose fermentation were achieved with more than 80% moisture content and the highest substrate conversion rate (21.9 mg L^?1 h^?1) was obtained with 90% moisture content at batch solid state fermentation producing volatile fatty acids (VFA) and H₂.     

Biohydrogen production by extracted fermentation from sugar beet

In the study, the production of biohydrogen by extracted fermentation from sugar beet was evaluated. Effects of initial amount of sugar beet, biomass and particle size of sugar beet on biohydrogen formation were investigated. The hydrogen (H₂) gas was predicted to be 78.6 mL at initial dry weight of sugar beet 24.6 g L^?1 and H₂ yield was calculated as 81.9 mLH₂ g^?1TOC while biomass concentration (1 g L^?1) and particle size (0.3 cm) were constant. The peak H₂ gas volume was predicted to be 139.9 mL at the low particle size of 0.1 cm. Hydrogen gas production potential was predicted as 143.6 mL h^?1. The peak value of 197.9 mLH₂ g^?1TOC was obtained with particle size of 0.1 cm when dry weight of sugar beet and initial amount of biomass was kept constant at 24.6 g L^?1 and 1 g L^?1, respectively.     

Effect of hydraulic retention time on a continuous biohydrogen production in a packed bed biofilm reactor with recirculation flow of the liquid phase

The present paper reports on results obtained from experiments carried out in a laboratory-scale anaerobic packed bed biofilm reactor (APBR), with recirculation of the liquid phase, for continuously biohydrogen production via dark fermentation. The reactor was filled with Kaldnes^® biofilm carrier and inoculated with an anaerobic mesophilic sludge from a urban wastewater treatment plant (WWTP). The APBR was operated at a temperature of 37 °C, without pH buffering. The effect of theoretical hydraulic retention time (HRT) from 1 to 5 h on hydrogen yield (HY), hydrogen production rate (HPR), substrate conversion and metabolic pathways was investigated. This study indicates the possibility of enhancing hydrogen production by using APBR with recirculation flow. Among respondents values of HRT the highest average values of HY (2.35 mol H₂/mol substrate) and HPR (0.085 L h^?1L^?1) have been obtained at HRT equal to 2 h.     

Evaluation of semi-continuous hydrogen production from enzymatic hydrolysates of Agave tequilana bagasse: Insight into the enzymatic cocktail effect over the co-production of methane

This work addresses the hydrogen production from enzymatic hydrolysates of Agave tequilana bagasse and the valorization of the acidogenic effluent for methane production in anaerobic sequencing batch reactors (ASBRs). Regarding hydrogen production, the ASBR was operated at four organic loading rates (OLRs), which were modified by decreasing the cycle time (from 24 to 12 h) and increasing the COD concentration (from 8 to 12 and 16 g L^?1). Results showed that the highest OLR promoted the highest hydrogen production rate of 25.2 ± 2.1 NmL L^?1 h^?1. Conversely, the hydrogen molar yield remained constant, obtaining similar values to the highest reported for lignocellulosic hydrolysates in continuous reactors (1.6H₂-mol mol_{consumed sugar}^?1). Regarding methane production from the acidogenic effluent, an unexpected methane suppression was observed during the first 5 cycles of the ASBR operation. Such event was attributed to the disaggregation of the granular sludge due to the remaining hydrolytic activity of the enzymatic cocktail used for the hydrolysates production. This was corroborated by feeding acetate to an ASBR (positive control) and supplying the enzymatic cocktail. Overall, even though the ASBR configuration demonstrated its suitability for hydrogen production, further studies are needed to coupling the methanogenic phase in different reactor configurations.     

Influence of C/P and C/N ratios and microbial characterization in hydrogen and ethanol production in an anaerobic fluidized bed reactor

The aim of this study was to evaluate the influence of C/P and C/N ratios on the production of hydrogen and ethanol in four anaerobic fluidized bed reactors: R1 (C/N = 100), R2 (C/N = 150), R3 (C/N = 200), and R4 (C/N = 250). The hydraulic retention time (HRT) was maintained at 2 h, and the C/P ratios varied from 300 to 1100. Reactors were filled with grounded tire and fed with synthetic substrate containing glucose (5000 mg L^?1). The effluent pH was around 3.7. The highest values for hydrogen yield (HY) and hydrogen production rate (HPR) were obtained at a C/P = 700 ratio in all reactors. The best performance was achieved at R3 (C/N = 200): HY of 0.76 mol H₂ mol^?1 glucose and HPR of 0.70 L h^?1 L^?1. The highest value for ethanol yield was obtained at C/P = 700 in R1 (1.5 mol EtOH mol^?1 glucose). Ethanol- and hydrogen-producing fermenters, such as Ethanoligenens sp. and Clostridium sp. were identified by molecular analysis. Lactobacillus sp. was also identified in this study.     

Bioethanol production from corn stalk juice using Saccharomyces cerevisiae TISTR 5020

This study aimed to use sweet corn hybrid hi-brix53 stalk juice for bioethanol production, to give a solution to the growing problem of food vs. fuel and to utilize waste for cheaper production. Hi-brix 53 stalk juice contained 112.07 ± 2.99 g L^?1 of total sugars and 21.83 ± 1.09 g L^?1 of reducing sugars. Through fermentation (24–120 h) using yeast (Saccharomyces cerevisiae), it produced 6.01% (v/v) bioethanol. The final ethanol produce (g L^?1) yield efficiency and volumetric ethanol productivity were at the highest at 24 h with 47.87 L^?1, 87.62% and 1.97 ± 0.06 (g L^?1 h^?1). These results suggest that hi-brix 53 stalk juice is an ideal substrate for bioethanol production.     

High hydrogen production rate in a submerged membrane anaerobic bioreactor

Batch cultivation of an anaerobic consortium fed with glucose as sole carbon source showed a sharp decrease of the hydrogen productivity when volatile fatty acids (VFA) concentration exceeded 12.5 g L^?1. To avoid VFA accumulation, fermentative batch cultures were thereafter carried out with a submerged membrane anaerobic bioreactor to continuously remove hydrogen fermentation co-products, while retaining the biomass. The membrane made it possible to separate the residence times of bacterial biomass and hydraulic part. With this technology, average and maximal productivities reached 0.75 and 2.46 L_H2 L^?1 h^?1, corresponding to an increase of 44 and 51% in comparison to the control, respectively. By removing the VFAs from the cultivation medium, H₂-producing pathways were favored, confirming the metabolic inhibitory effects of co-product accumulation in fermentation medium. Such hydrogen productivity is one of the highest values encountered in the literature. Readily implementable, such technology offers a good opportunity for further developing high rate hydrogen fermentation bioprocesses.     

Photohydrogen production from lactose and lactate by recombinant strains of Rhodobacter capsulatus: Modeling and optimization

Photofermentative hydrogen production from synthetic mixtures of lactose and lactate mimicking cheese whey was modeled and optimized using Design of Experiments and Response Surface Methodology. Five continuous parameters (light intensity, pH, lactose, lactate and glutamate concentrations) were studied as a function of buffer type (KPi or Borax) using two recombinant bacterial strains. For Rhodobacter capsulatus B10(lacZ), buffer type influenced the optimal parameter values but the optimal responses were similar in both buffers. In contrast, for R. capsulatus IR3(lacZ), responses were higher in Borax buffer than in KPi and were significantly higher than in strain B10(lacZ). Thus, the experimental optimized responses for specific volumetric H₂ production, volumetric H₂ production rate and substrate (lactose plus lactate) to H₂ conversion rate in Borax buffer, were 12,150 ml L^?1, 48.5 ml L^?1 h^?1 and 41.2%, respectively, for IR3(lacZ) compared to 6150 ml L^?1, 33.5 ml L^?1 h^?1 and 32.5%, respectively, for B10(lacZ).     

Prolongation of H2 production during mixed carbon sources fermentation in E. coli batch cultures: New findings and role of different hydrogenases

Hydrogen (H₂) gas production in batch cultures was studied upon utilization of the mixture of glucose, glycerol and formic acid by Escherichia coli BW25113 wild type (wt) at pH of 5.5–7.5. At pH 7.5H₂ was continuously produced during 240 h but at pH 6.5 and 5.5 it was detected till 168 h and 120 h, respectively. Specific growth rate (μ) of wt was the highest (1.05 h^?1) at pH 6.5. Moreover, at pH 5.5 in hycE μ decreased by ～4.14 fold compared to wt, suggesting major role of Hyd-3 in cell growth. H₂ yield (8.8 mmol H₂ L^?1) was the highest at pH 7.5. In hybC H₂ yield was increased ～1.62 fold than in wt. These data might be applied for biomass and biohydrogen production from various organic wastes where mixtures of carbon sources are present.     

Assessment of the adequacy of different Mediterranean waste biomass types for fermentative hydrogen production and the particular advantage of carob (Ceratonia siliqua L.) pulp

The conversion of agro-industrial byproducts, residues and microalgae, which are representative or adapted to the Mediterranean climate, to hydrogen (H₂) by C. butyricum was compared. Five biomass types were selected: brewery’s spent grain (BSG), corn cobs (CC), carob pulp (CP), Spirogyra sp. (SP) and wheat straw (WS). The biomasses were delignified and/or saccharified, except for CP which was simply submitted to aqueous extraction, to obtain fermentable solutions with 56.2–168.4 g total sugars L^?1. In small-scale comparative assays, the H₂ production from SP, WS, CC, BSG and CP reached 37.3, 82.6, 126.5, 175.7 and 215.8 mL (g biomass)^?1, respectively. The best fermentable substrate (CP) was tested in a pH-controlled batch fermentation. The H₂ production rate was 204 mL (L h)^?1 and a cumulative value of 3.9 L H₂ L^?1 was achieved, corresponding to a H₂ production yield of 70.0 mL (g biomass)^?1 or 1.6 mol (mol of glucose equivalents)^?1_. The experimental data were used to foresight a potential energy generation of 2.4 GWh per year in Portugal, from the use of CP as substrate for H₂ production.     

Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation

This article describes the ability of the Chlorella vulgaris BEIJ strain G-120 to produce hydrogen (H₂) via both direct and indirect pathways without the use of nutrient starvation. Photobiological H₂ production reached a maximum rate of 12 mL H₂ L^?1 h^?1, corresponding to a light conversion efficiency (light to H₂) of 7.7% (average 3.2%, over the 8-day period) of PAR, (photosynthetically active irradiance). Cells presented a maximum in vivo hydrogenase activity of 25.5 ± 0.2 nmoles H₂ μgChl^?1 h^?1 and the calculated in vitro hydrogenase activity was 830 ± 61 nmoles H₂ μgChl^?1 h^?1. The strain is able to grow either heterotrophically or photo autotrophically. The total output of 896 mL of H₂ was attained for illuminated culture and 405 mL for dark cultures. The average H₂ production rate was 4.98 mL L^?1 h^?1 for the illuminated culture and 2.08 mL L^?1 h^?1 for the one maintained in the dark.     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.     

Production of biohydrogen from brewery wastewater using Klebsiella pneumoniae isolated from the environment

The use of wastewater for the biological production of H₂ (biohydrogen) by dark fermentation has been studied for a variety of waste substrates and mixed or isolated inocula. However, for brewery wastewater (BW), which is generated in large volumes and has characteristics that are highly suitable for acidogenic fermentation, the available studies describe the use of mixed cultures, especially pretreated methanogenic inocula. The aim of this work was to isolate an enterobacterium from aviary litter that was capable of fermenting BW and generating biogas rich in H₂. The biochemical characterization and species confirmation confirmation revealed the isolation of Klebsiella peneumoniae, which provided efficient production of biogas rich in H₂ (30–40%) in batch assays performed for up to 72 h, with the inoculum in suspension, at a small scale (in serum bottles) and using a mechanically-stirred anaerobic reactor (AnBBR), employing crude BW without any supplementation. The hydrogen yield and molar hydrogen flow rate were 0.80–1.67 mol H₂ mol^?1 glucose and 0.2–2.2 mmol H₂ h^?1, respectively, indicating good performance of the inoculum in metabolizing this substrate and the possibility of optimizing the process by varying the duration of the batch.