Enhancing thermophilic dark fermentative hydrogen production at high glucose concentrations via bioaugmentation with Thermotoga neapolitana

The aim of the present study was to investigate the effect of gradually increasing glucose concentrations (from 5.6 to 111 mmol L⁻¹) on the fermentative H₂ production with and without bioaugmentation. A stirred tank reactor (STR) was operated at 70 °C and inoculated with a hyperthermophilic mixed culture or a hyperthermophilic mixed culture bioaugmented with Thermotoga neapolitana. With both the unaugmented (control) and augmented cultures, the H₂ production rate was improved when the initial glucose concentration was increased. In contrast, the highest H₂ yield (1.68 mol H₂ mol⁻¹ glucose consumed) was obtained with the augmented culture at the lowest glucose concentration of 5.6 mmol L⁻¹ and was 37.5% higher than that obtained with the unaugmented culture at the same feed glucose concentration. Overall, H₂ production rates and yields were higher in the bioaugmented cultures than in the unaugmented cultures whatever the glucose concentration. Quantitative polymerase chain reaction targeting T. neapolitana hydA gene and MiSeq sequencing proved that Thermotoga was not only present in the augmented cultures but also the most abundant at the highest glucose concentrations.     

Biomass and biohydrogen production during dark fermentation of Escherichia coli using office paper waste and cardboard

Growth properties, oxidation-reduction potential kinetics and hydrogen production of Escherichia coli BW25113 parental strain (PS) and hydrogenase (Hyd)-negative mutants were investigated after fermentative growth using office paper waste and cardboard (PW) hydrolysate (PWH). PWH was obtained by using dilute acid method in a steam sterilizer for 1 h, 121 °C. Optimal conditions for bacterial growth and H₂ production were identified (PWH concentrations, pH 7.5). Recording of redox potential using a platinum electrode revealed a drop to −500 ± 10 mV, with a H₂ yield of ~1.45 mmol H₂ L⁻¹ after 4 h of growth using PWH resulted in the formation of 0.20 ± 0.02 g bacterial cell dry weight L⁻¹. Bacterial biomass formation was stimulated ~3-fold upon addition of 0.5% yeast extract, and H₂ production started early - at the beginning of the exponential phase. Moreover, mutants lacking Hyd-1 and Hyd-2 significantly enhanced H₂ production. The findings would be beneficial for the development of H₂ production biotechnology using cheap solid waste materials.     

Ozonation aided mesophilic biohydrogen production from palm oil mill effluent

Ozone pretreatment of palm oil mill effluent (POME) was employed to improve sustrate biodegradability prior to biological H₂ production. The H₂ production was conducted at varing pHs from 4.0 to 6.0 to examine the impact of pH on the H₂ mesophilic production (37 °C). The optimal pH for H₂ production was 6.0 for both raw and ozonated POME. The POME concentrations were greatly influenced the yields and rates of H₂ production. At the optimal pH, the maximum H₂ production yield of 182 ± 7.2 mL.g⁻¹ COD (7.96 mmoL.g⁻¹ COD) was achieved at the ozonated POME concentration of 30,000 mg COD.L⁻¹. The maximum H₂ production rate (R_max) of 43.1 ± 2.5 mL.h⁻¹ was obtained at the ozonated POME concentration of 25,000 mg COD.L⁻¹. The highest total COD removal was 44% at of 15,000 mg COD.L⁻¹ ozonated POME. Acetic and butyric acids were dominant products during H₂ fermentation and tended to increase with the increased POME concentrations. Ozonation as a pretreatment process showed significant enhancement of the POME biodegradability , and subsequently improved the H₂ production H₂.     

Thermodynamic and kinetic properties of calcium hydride

Calcium hydride has shown great potential as a hydrogen storage material and as a thermochemical energy storage material. To date, its high operating temperature (above 800 °C) has not only hindered its opportunity for technological application but also prevented detailed determination of its thermodynamics of hydrogen sorption. In addition, calcium metal suffers from high volatility, high corrosivity from Ca (and CaH₂), slow kinetics of hydrogen sorption, and the solubility of Ca in CaH₂. In this work, a literature review of the wide-ranging thermodynamic properties of CaH₂ is provided along with a detailed experimental investigation into the thermodynamic properties of molten and solid CaH₂. The thermodynamic values of hydrogen release from both molten and solid CaH₂ were determined as ΔH_des (molten CaH₂) = 216 ± 10 kJ mol⁻¹.H₂, ΔS_des (molten CaH₂) = 177 ± 9 J K⁻¹ mol⁻¹.H_2, which equates to a 1 bar hydrogen equilibrium temperature for molten CaH₂ of 947 ± 65 °C. Similarly, in the solid-state: ΔH_des (solid CaH₂) = 172 ± 12 kJ mol⁻¹.H₂, ΔS_des (solid CaH₂) = 144 ± 10 J K⁻¹ mol⁻¹.H₂. Moreover, the activation energy of hydrogen release from CaH₂ was also calculated using DSC analysis as E_a = 203 ± 12 kJ mol⁻¹. This study provides the first thermodynamics for the Ca–H system in over 60 years, providing more accurate data on this emerging energy storage material.     

Thermophilic hydrogen and methane production from sugarcane stillage in two-stage anaerobic fluidized bed reactors

This study evaluated the feasibility of H₂ and CH₄ production in two-stage thermophilic (55 °C) anaerobic digestion of sugarcane stillage (5,000 to 10,000 mg COD.L⁻¹) using an acidogenic anaerobic fluidized bed reactor (AFBR-A) with a hydraulic retention time (HRT) of 4 h and a methanogenic AFBR (AFBR-S) with HRTs of 24 h–10 h. To compare two-stage digestion with single-stage digestion, a third methanogenic reactor (AFBR-M) with a HRT of 24 h was fed with increasing stillage concentrations (5,000 to 10,000 mg COD.L⁻¹). The AFBR-M produced a methane content of 68.4 ± 7.2%, a maximum yield of 0.30 ± 0.04 L CH₄.g COD⁻¹, a production rate of 3.78 ± 0.40 L CH₄.day⁻¹.L⁻¹ and a COD removal of 73.2 ± 5.0% at an organic loading rate (OLR) of 7.5 kg COD.m⁻³.day⁻¹. In contrast, the two-stage AFBR-A system produced a hydrogen content of 23.9 ± 5.6%, a production rate of 1.30 ± 0.16 L H₂.day⁻¹.L⁻¹ and a yield of 0.34 ± 0.08 mmol H₂.g CODap⁻¹. Additionally, the decrease in the HRT from 18 h to 10 h in the AFBR-S favored a higher methane production, improving the maximum methane content (74.5 ± 6.0%), production rate (5.57 ± 0.38 L CH₄.day⁻¹.L⁻¹) and yield (0.26 ± 0.06 L CH₄.g COD⁻¹) at an OLR of 21.6 kg COD.m⁻³.day⁻¹ (HRT of 10 h) with a total COD removal of 70.1 ± 7.1%. Under the applied COD of 10,000 mg L⁻¹, the two-stage system showed a 52.8% higher energy yield than the single-stage anaerobic digestion system. These results show that, relative to a single-stage system, two-stage anaerobic digestion systems produce more hydrogen and methane while achieving similar treatment efficiencies.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Chitosan flocculation associated with biofilms of C. saccharolyticus and C. owensensis enhances biomass retention in a CSTR

Cell immobilization and co-culture techniques have gained attention due to its potential to obtain high volumetric hydrogen productivities (Q_H2). Chitosan retained biomass in the fermentation of co-cultures of Caldicellulosiruptor saccharolyticus and C. owensensis efficiently, up to a maximum dilution rate (D) of 0.9 h^?1. Without chitosan, wash out of the co-culture occurred earlier, accompanied with approximately 50% drop in Q_H2 (D > 0.4 h^?1). However, butyl rubber did not show as much potential as carrier material; it did neither improve Q_H2 nor biomass retention in continuous culture. The population dynamics revealed that C. owensensis was the dominant species (95%) in the presence of chitosan, whereas C. saccharolyticus was the predominant (99%) during cultivation without chitosan. In contrast, the co-culture with rubber as carrier maintained the relative population ratios around 1:1. This study highlighted chitosan as an effective potential carrier for immobilization, thereby paving the way for cost – effective hydrogen production.     

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of Thermotoga maritima

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H₂) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H₂ production limit. The study generated a kinetic model explaining H₂ overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H₂ production by Tma200 (0.217 mol-H₂ g⁻¹-biomass) and Tma100 (0.147 mol-H₂ g⁻¹-biomass) were higher than wild type (0.096 mol-H₂ g⁻¹ -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h⁻¹) and Tma100 (0.495 h⁻¹) are compared with the wild type (0.663 h⁻¹). A higher maintenance energy along with growth and non-growth H₂ coefficients corroborate the higher H₂ productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H₂ production.     

Hydrogen metabolism in the extreme thermophile Thermotoga neapolitana

Anaerobic growth of Thermotoga neapolitana led maximum to hydrogen yield of 3.85 ± 0.07 mol H₂/mol glucose and production rate of 51 ml/l/h. This productivity is strongly affected by stirring, pH buffering, N₂ sparging and culture/headspace volume ratio. Embden–Meyerhoff pathway is the only glycolytic route in T. neapolitana but, under the conditions used in this study, about 12–15% of the biogas requires consumption of protein source. Reduction of the hydrogen yields below the theoretical 4 mol H₂/mol glucose is mainly due to production of lactate and alanine that affect the availability of pyruvate/NADH for the hydrogenase, as well as to loss of part of glucose by conversion to fructose that is eventually released in the medium. Hydrogen productivity is modulated during the bacterial growth and major biogas synthesis is recorded in the stationary phase in concomitance with reduction of lactate synthesis. Apparently, this event is not consistent with an equal increase in acetate production. In agreement with the hydrogenase model recently proposed for the sister species Thermotoga maritima, this suggests that cellular NADH⁺ ratio has a crucial role on biogas synthesis.     

Modeling the kinetic behavior of the Li-RHC system for energy-hydrogen storage: (I) absorption

The Lithium–Boron Reactive Hydride Composite System (Li-RHC) (2 LiH + MgB₂/2 LiBH₄ + MgH₂) is a high-temperature hydrogen storage material suitable for energy storage applications. Herein, a comprehensive gas-solid kinetic model for hydrogenation is developed. Based on thermodynamic measurements under absorption conditions, the system's enthalpy ΔH and entropy ΔS are determined to amount to −34 ± 2 kJ∙mol H₂⁻¹ and −70 ± 3 J∙K⁻¹∙mol H₂⁻¹, respectively. Based on the thermodynamic behavior assessment, the kinetic measurements' conditions are set in the range between 325 °C and 412 °C, as well as between 15 bar and 50 bar. The kinetic analysis shows that the hydrogenation rate-limiting-step is related to a one-dimensional interface-controlled reaction with a driving-force-corrected apparent activation energy of 146 ± 3 kJ∙mol H₂⁻¹. Applying the kinetic model, the dependence of the reaction rate constant as a function of pressure and temperature is calculated, allowing the design of optimized hydrogen/energy storage vessels via finite element method (FEM) simulations.     

Inhibition by the ionic strength of hydrogen production from the organic fraction of municipal solid waste

Composition of the Organic Fraction of Municipal Solid Waste (OFMSW) in organic compounds and inorganic ions is highly variable and might impact the microbial activity in dark fermentation processes. In this study, the effect of the total amount of inorganic ions on fermentative hydrogen production was investigated. Batch experiments were carried out at pH 6 and under a temperature of 37 °C. A freshly reconstituted organic fraction of municipal solid waste (OFMSW) was used as model substrate. At low concentrations in ammonium or chloride ions (2.9–5.1 g L⁻¹, respectively), the hydrogen yield reached a maximum of 40.8 ± 0.5. mLH₂.gVS⁻¹ and 25.1 ± 5.6 mLH₂.gVS⁻¹. In contrast, at high total ionic concentrations of ammonium and chloride (11.1–35.5 g L⁻¹ respectively), a strong inhibition of the fermentative microbial activity and more particularly hydrogen production, was observed. When considering the ionic strength of each ion, the effects of ammonia, chloride or a mixture of different ions (Na⁺, K⁺, H⁺, Li⁺, NH₄⁺, Mn²⁺, NH₄⁺, Mg²⁺, Cl⁻, PO₄³⁻, Br⁻, I⁻, SO₄²⁻) showed very similar inhibitory trends regardless the type of ion or the composition of the ionic mixture. A threshold inhibitory value of the ionic strength was estimated at 0.75 ± 0.13 M with a substantial impact on the fermentative activity from 0.81 ± 0.12 M, with hydrogen yields of 18.1 ± 3.3 and 6.2 ± 4.1 mLH₂.gVS⁻¹, respectively. Microbial community composition was also significantly impacted with a specific decrease in relative abundance of hydrogen-producing bacteria from the genus Clostridium sp. at high ionic strength.     

Hydrogen production in reactors: The influence of organic loading rate,inoculum and support material

Hydrogen production was evaluated in two thermophilic structured bed (USBR) reactors. USBR1was inoculated with auto-fermented sugarcane vinasse and low-density polyethylene cubes were used as support material. USBR2 was inoculated with anaerobic sludge from an up-flow anaerobic sludge blanket (UASB) reactor treating sugarcane vinasse, and polyurethane foam matrices was used as support material. The reactors were operated in parallel with sugar cane molasses at organic loading rate (OLR) from 30 to 120 g COD L⁻¹d⁻¹ during 45 days. Hydrogen production was detected during the whole operational period, with maximum values of 1123 mL H₂ d⁻¹L⁻¹ and 2041 mL H₂ d⁻¹L⁻¹ for USBR1 and USBR2, respectively. The number of gene copies encoding for Fe-hydrogenase was higher in USBR2 for all OLR applied. DNA sequences related to Thermoanaerobacterium and Clostridium sensu stricto were predominant in USBR1. In USBR2, in addition to these microorganisms, Lactobacillus, Pseudomonas and Thermotuga, and sequences with low frequency of abundance (<5%) involved directly and indirectly in hydrogen production were also present. The taxonomical and functional more diverse inoculum of USBR2 was associated with a higher hydrogen production. Besides fermentation, an unknown metabolism was relevant in USBR2, revealing the importance of physiological characterization of the microbial community present.     

Impacts of short-term temperature fluctuations on biohydrogen production and resilience of thermophilic microbial communities

Anaerobic microflora enriched for dark fermentative H₂ production from a mixture of glucose and xylose was used in batch cultivations to determine the effects of sudden short-term temperature fluctuations on H₂ yield and microbial community composition. Batch cultures initially cultivated at 55 °C (control) were subjected to downward (from 55 °C to 35 °C or 45 °C) or upward (from 55 °C to 65 °C or 75 °C) temperature shifts for 48 h after which, each culture was transferred to a fresh medium and cultivated again at 55 °C for two consecutive batch cycles. The average H₂ yield obtained during the first cultivation at 55 °C was 2.1 ± 0.14 mol H₂ mol⁻¹ hexose equivalent. During the temperature shifts, the obtained H₂ yields were 1.8 ± 0.15, 1.6 ± 0.27 and 1.9 ± 0.00 mol H₂ mol⁻¹ hexose equivalent at 35 °C, 45 °C and 65 °C, respectively, while no metabolic activity was observed at 75 °C. The sugars were completely utilized during the 48 h temperature shift to 35 °C but not at 65 °C and 45 °C. At the end of the second cycle after the different temperature shifts, the H₂ yield obtained was 96.5, 91.6, 79.9 and 54.1% (second cycle after temperature shift to 35 °C, 45 °C, 65 °C and 75 °C, respectively) when compared to the average H₂ yield produced in the control at 55 °C. Characterization of the microbial communities present in the control culture at 55 °C showed the predominance of Thermoanaerobacteriales, Clostridiales and Bacilliales. The microbial community composition differed based on the fluctuation temperature with Thermoanaerobacteriales being most dominant during the upward temperature fluctuations and Clostridiales being the most dominant during the downward temperature fluctuations.     

Comparative evaluation of the mesophilic and thermophilic biohydrogen production at optimized conditions using tequila vinasses as substrate

The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H₂) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 °C and 55 °C, and pH of 5.5 maximized the bio-H₂ production rates, 25.5 ± 0.01 NmL h⁻¹ and 169.9 ± 8.9 NmL h⁻¹ in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H₂ production rate of 519 ± 13 NmL-H₂ L⁻¹ d⁻¹ equivalent to 750 ± 19 NmL-H₂ L_vinasse⁻¹, while the mesophilic one 448 ± 23 NmL-H₂ L⁻¹ d⁻¹ and 647 ± 33 NmL-H₂ L_vinasse⁻¹, respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H₂ production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed.     

Extreme thermophilic condition: An alternative for long-term biohydrogen production from sugarcane vinasse

Boosted by the high temperatures in which vinasse is generated (90 °C–100 °C), this study evaluated the effect of an extreme thermophilic condition (70 °C) on sugarcane vinasse Dark Fermentation (DF) in an Anaerobic Structured Bed Reactor (ASTBR). Four hydraulic retention times (HRT) (19, 15, 12 and 8 h) were evaluated. Higher HRT resulted in a greater H₂ production rate (690 mLH₂.d⁻¹.L⁻¹), higher yields (1.8 molH₂.mol_Glucose⁻¹) and greater stability. The extreme temperature inhibits microorganisms' extracellular polymer production, thus leading to a disperse growth, preventing excess biomass accumulation, which was previously reported as the main drawback in H₂ production at lower temperatures. The ASTBR higher void index is also responsible for lower biomass/solids retention. The H₂ production main route was through the lactic/acetic acid pathway, which is highly reliant on the pH of fermentation broth. The main genus involved in H₂ production at 70 °C were Clostridium, Pectinatus, Megasphaera and Lactobacillus.     

Screening and optimization of trace elements supplement in sweet sorghum juice for ethanol production

Eight trace elements were screened for increasing efficiency of ethanol yield from sweet sorghum juice using the Plackett-Burman design method. MnCl₂·4H₂O, CoCl₂·6H₂O and biotin was screened as the significant variables which have positive effects on ethanol production from sweet sorghum juice. The values of MnCl₂·4H₂O, CoCl₂·6H₂O and biotin, optimized by Box-Behnken design method, were 7.70 mg L⁻¹, 15.74 mg L⁻¹ and 11.97 mg L⁻¹, respectively. The experimental efficiency of ethanol yield under optimal conditions was 89.30 ± 0.10%, which enhances the efficiency of ethanol yield by 5.63% by the addition of MnCl₂·4H₂O, CoCl₂·6H₂O and biotin. The results from this study have identified optimal conditions as a foundation for pilot scale ethanol production.     

Fermentative hydrogen production from cheese whey with in-line,concentration gradient-driven butyric acid extraction

Hydrogen (H₂) generation from cheese whey with simultaneous production and extraction of volatile fatty acids (VFAs) was studied in UASB reactors at two temperatures (20 and 35 °C) and pH values (5.0 and 4.5). The extraction module, installed through a recirculation loop, was a silicone tube coil submerged in water, which allows concentration-driven extraction of undissociated VFAs. Operating conditions were selected as a compromise for the recovery of both H₂ and VFAs. Batch experiments showed a higher yield (0.9 mol H₂ mol⁻¹ glucose_eq.) at 35 °C and pH 5.0, regardless of the presence of the extraction module, whereas lower yields were obtained at pH 4.5 and 20 °C (0.5 and 0.3 mol H₂ mol⁻¹ glucose_eq., respectively). VFAs crossed the silicone membrane, with a strong preference for butyric over propionic and acetic acid due to its higher hydrophobicity. Sugars, lactic acid and nutrients were retained, resulting in an extracted solution of up to 2.5 g L⁻¹ butyric acid with more than 90% purity. Continuous experiment confirmed those results, with production rates up to 2.0 L H₂ L⁻¹ d⁻¹ and butyric acid extraction both in-line (from the UASB recirculation) and off-line (from the UASB effluent). In-line VFA extraction can reduce the operating costs of fermentation, facilitating downstream processing for the recovery of marketable VFAs without affecting the H₂ production.     

Coproduction of hydrogen,ethanol and 2,3-butanediol from agro-industrial residues by the Antarctic psychrophilic GA0F bacterium

In this study, the simultaneous production of hydrogen, ethanol, and 2,3-butanediol was assessed using three agro-industrial residues: cheese whey powder (CWP), wheat straw hydrolysate (WSH) and sugarcane molasses (SCM), by the Antarctic psychrophilic GA0F bacterium [EU636050], which is closely related to Pseudomonas antarctica [KX186936.1]. The main soluble metabolites produced in all the fermentations were ethanol and 2,3-butanediol. CWP demonstrated to be the most effective carbon source, since fermentation of this substrate resulted in the highest yields of H₂ (73.5 ± 10 cm³ g⁻¹), ethanol (0.24 ± 0.03 g g⁻¹) and 2,3-butanediol (0.42 ± 0.04 g g⁻¹), followed by the use of SCM, whereas WSH showed to have an inhibitory effect during the fermentation process, showing the lowest production values. Our results demonstrated the ability of the Antarctic psychrophilic GA0F bacterium to produce valuable products using low-cost substrates at room temperature conditions.