Enhanced Biohydrogen Production Research Articles

Enhancing Biohydrogen Production: The Role of Iron-Based Nanoparticles in Continuous Lactate-Driven Dark Fermentation of Powdered Cheese Whey

Here, a comprehensive investigation was conducted under various operational strategies aimed at enhancing biohydrogen production via dark fermentation, with a specific focus on the lactate metabolic pathway, using powdered cheese whey as a substrate. Initially, a batch configuration was tested to determine both the maximum hydrogen yield (100.2 ± 4.2 NmL H2/g CODfed) and the substrate (total carbohydrates) consumption efficiency (94.4 ± 0.8%). Subsequently, a transition to continuous operation was made by testing five different operational phases: control (I), incorporation of an inert support medium for biomass fixation (II), addition of carbon-coated, zero-valent iron nanoparticles (CC-nZVI NPs) at 100 mg/L (III), and supplementation of Fe2O3 nanoparticles at concentrations of 100 mg/L (IV) and 300 mg/L (V). The results emphasized the critical role of the support medium in stabilizing the continuous system. On the other hand, a remarkable increase of 10% in hydrogen productivity was observed with the addition of Fe2O3 NPs (300 mg/L). The analysis of the organic acids’ composition unveiled a positive correlation between high butyrate concentrations and improved volumetric hydrogen production rates (25 L H2/L-d). Moreover, the presence of iron-based NPs effectively regulated the lactate concentration, maintaining it at low levels. Further exploration of the bacterial community dynamics revealed a mutually beneficial interaction between lactic acid bacteria (LAB) and hydrogen-producing bacteria (HPB) throughout the experimental process, with Prevotella, Clostridium, and Lactobacillus emerging as the predominant genera. In conclusion, this study highlighted the promising potential of nanoparticle addition as a tool for boosting biohydrogen productivity via lactate-driven dark fermentation.

Comparative Analysis of Various Seed Sludges for Biohydrogen Production from Alkaline Pretreated Rice Straw

The present work studied the effects of alkali pretreatment on the cellulosic biomass of rice straw. The improvement in the cellulose content and reduction in the lignin and hemicellulose percentage was observed with alkali pretreatment. Fourier transformation infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM) analysis confirm the modification in the surface structure of alkali rice straw. Further, the study investigated the potential of different types of seed sludge as inoculum sources for dark fermentative biohydrogen production. In comparison to other sludge samples (beverage industry, food industry, and sewage treatment plant sludge), the mixed culture of sewage treatment plant sludge had the highest cumulative volume of biohydrogen (90.52 mL), as well as the highest hydrogen production yield (0.75 moleH2/mole) with the substrate utilization of 86.72%. The results provide information on the best sludge source for enhancing biohydrogen production in the dark fermentation method.

Improved biohydrogen production by co-fermentation of corn straw and excess sludge: Insights into biochemical process, microbial community and metabolic genes

The global climate change mainly caused by fossil fuels combustion promotes that zero-carbon hydrogen production through eco-friendly methods has attracted attention in recent years. This investigation explored the biohydrogen production by co-fermentation of corn straw (CS) and excess sludge (ES), as well as comprehensively analyzed the internal mechanism. The results showed that the optimal ratio of CS to ES was 9:1 (TS) with the biohydrogen yield of 101.8 mL/g VS, which was higher than that from the mono-fermentation of CS by 1.0-fold. The pattern of volatile fatty acids (VFAs) indicated that the acetate was the most preponderant by-product in all fermentation systems during the biohydrogen production process, and its yield was improved by adding appropriate dosage of ES. In addition, the content of soluble COD (SCOD) was reduced as increasing ES, while concentration of NH4+-N showed an opposite tendency. Microbial community analysis revealed that the microbial composition in different samples showed a significant divergence. Trichococcus was the most dominant bacterial genus in the optimal ratio of 9:1 (CS/ES) fermentation system and its abundance was as high as 41.8%. The functional genes prediction found that the dominant metabolic genes and hydrogen-producing related genes had not been significantly increased in co-fermentation system (CS/ES = 9:1) compared to that in the mono-fermentation of CS, implying that enhancement of biohydrogen production by adding ES mainly relied on balancing nutrients and adjusting microbial community in this study. Further redundancy analysis (RDA) confirmed that biohydrogen yield was closely correlated with the enrichment of Trichococcus.

Enhanced bio-hydrogen production by photo-fermentation of corn stalk using Fe-doped CaTiO3 photocatalyst

The aim of this study was to analyze the effects of CaTiO3 photocatalysts doped with different concentrations of Fe on the biohydrogen production process by photo-fermentation using corn stover as the substrate. The experimental results showed that the maximum cumulative hydrogen production and the maximum rate of hydrogen production were obtained when the doping concentration was 6 % (g/g) (380.8 mL, 26.4 mL/h), which were 63.4 % and 203.4 % higher, respectively, than those of the blank control group (233 mL, 8.7 mL/h). The substrate conversion rate was 32.82 %. Iron-doped CaTiO3 lowered the oxygen reduction potential in the fermentation system to provide a suitable reduction environment for the growth and fermentation of photosynthetic bacteria. The lowest redox potential observed was −393.5 mV for 6 % iron-doped CaTiO3. Thermogram analysis revealed a strong correlation between cumulative hydrogen production and redox potential. Additionally, the analysis of soluble small molecules showed that iron-doped CaTiO3 could inhibit the production of ethanol from glycolysis during fermentation and promote the utilization of small molecule acids.

Screening of enhanced biohydrogen production from anaerobic fermentation amended with nano-sized barium ferrite supported by aluminum oxide

Anaerobic fermentation has attracted wide attention to effectively reduce carbon footprint and alleviate potential energy shortages, which is being regarded as a sustainable and promising technology to obtain high biohydrogen production from widespread organic wastes. However, biohydrogen commercial uptake is now dominantly inhibited by the low hydrogen yield and process instability. In this work, Al2O3/BaFe2O4 nanoparticles (NPs) are successfully applied to anaerobic fermentation for investigating their effects on hydrogen yield, activity of bacteria and microbial community structure. The benefits of Al2O3/BaFe2O4 NPs with respect to microbial metabolism, reactivity of enzymes and process optimization are systematically researched through batch experiments. In addition, various characterizations are used to verify the physical and chemical properties of Al2O3/BaFe2O4 NPs. The results show that the activity of hydrogenases and microbe metabolism can be significantly inhibited by excess addition of Al2O3/BaFe2O4 NPs (150 mg/L and 200 mg/L) while the optimum addition 100 mg/L of Al2O3/BaFe2O4 NPs can boost the biohydrogen yield by 49% via facilitating microorganism growth and enhancing key enzymatic activities, which indicate that the Al2O3/BaFe2O4 NPs display a beneficial influence on electron transfer efficiency and process stability. The existence of Fe3+, Al3+and Ba2+ derived from the corrosion of Al2O3/BaFe2O4 NPs by organic acids synergistically improve the microbial transmembrane transportation and substrate utilization, which contribute to higher conversion rate of glucose into acetate, butyrate and H2. Meanwhile, the complementary functions of Fe3+ and Al3+ can powerfully shorten the lag phase and promote the glycolysis, which finally accelerate the dark fermentation process. Compared with the control group, Clostridium_sensu_stricto_1 is selectively enriched by the addition of Al2O3/BaFe2O4 NPs, which keep heavily involved in the synthesis of H2. On balance, this study provides an effective strategy to enhance hydrogen productivity and maintain process stability, which offer the valuable information for the sustainable utilization of sewage sludge.

Enhancing biohydrogen production from xylose through natural FeS2 ore: Mechanistic insights

In this study, we investigated the advantages of utilizing natural FeS2 ore in the context of dark fermentative hydrogen production within a fermentation system employing heat-treated anaerobic granular sludge with xylose as the carbon source. The results demonstrated a significant improvement in both hydrogen production and the maximum rate, with increases of 2.58 and 4.2 times, respectively. Moreover, the presence of FeS2 ore led to a reduction in lag time by more than 2–3 h. The enhanced biohydrogen production performance was attributed to factors such as the intracellular NADH/NAD+ ratio, redox-active components of extracellular polymeric substances, secreted flavins, as well as the presence of hydrogenase and nitrogenase. Furthermore, the FeS2 ore served as a direct electron donor and acceptor during biohydrogen production. This study shed light on the underlying mechanisms contributing to the improved performance of biohydrogen production from xylose during dark fermentation through the supplementation of natural FeS2 ore.

Strategies to enhance biohydrogen production from microalgae: A comprehensive review

Microalgae represent a promising renewable feedstock for the sustainable production of biohydrogen. Their high growth rates and ability to fix carbon utilizing just sunlight, water, and nutrients make them well-suited for this application. Recent advancements have focused on improving microalgal hydrogen yields and cultivation methods. This review aims to summarize recent developments in microalgal cultivation techniques and genetic engineering strategies for enhanced biohydrogen production. Specific areas of focus include novel microalgal species selection, immobilization methods, integrated hybrid systems, and metabolic engineering. Studies related to microalgal strain selection, cultivation methods, metabolic engineering, and genetic manipulations were compiled and analyzed. Promising microalgal species with high hydrogen production capabilities such as Synechocystis sp., Anabaena variabilis, and Chlamydomonas reinhardtii have been identified. Immobilization techniques like encapsulation in alginate and integration with dark fermentation have led to improved hydrogen yields. Metabolic engineering through modulation of hydrogenase activity and photosynthetic pathways shows potential for enhanced biohydrogen productivity. Considerable progress has been made in developing microalgal systems for biohydrogen. However, challenges around process optimization and scale-up remain. Future work involving metabolic modeling, photobioreactor design, and genetic engineering of electron transfer pathways could help realize the full potential of this renewable technology.

Effect of pH, temperature and agitation on thermophilic biohydrogen production using immobilized cells on carbon composites (GAC-NiFe3O4)

Carbon composites-immobilized cell was utilized to enhance biohydrogen production. The initial pH, temperature and agitation effect was studied using the one-factor-at-a-time (OFAT) method. The optimal initial pH obtained was at pH 6.0 with hydrogen yield (HY) of 2.66 ± 0.09 mol H2/mol sugar, correlating with Gompertz constant of Hm = 974.99 mL, Rm = 17.90 mL/h and λ = 1.70 h. At 60 °C, the highest HY was obtained at 2.75 ± 0.11 mol H2/mol sugar. These corresponded to the Gompertz constant of Hm = 934.58 mL, Rm = 18.78 mL/h and λ = 1.89 h. The optimal agitation obtained was at 120 rpm, attaining the highest HY of 3.44 ± 0.54 mol H2/mol sugar, corresponding to the Gompertz constant of Hm = 1054.86 mL, Rm = 24.37 mL/h and λ = 3.90 h. The rRNA sequencing result revealed that the predominant species in the study was Thermoanaerobacterium. This study provides critical insight into process control conditions on biohydrogen production.

Enhancement of biohydrogen production by photo-fermentation of corn stover via visible light catalyzed titanium dioxide/activated carbon fiber

In this study, titanium dioxide/activated carbon fiber (TiO2/ACF) was synthesized by liquid-phase deposition method and the effect of TiO2/ACF on the performance of photo-fermentation biohydrogen production (PFHP) from corn stover under visible light catalysis was discussed. Results show the maximum cumulative hydrogen yield (CHY) obtained under the optimal conditions was 74.0 ± 1.3 mL/g TS with TiO2/ACF addition of 100 mg/L, which was twice that without TiO2/ACF addition (36.9 ± 1.0 mL/g TS). Initial pH value had the most significant effect on CHY. The addition of TiO2/ACF promoted the metabolic pathway of nitrogenase to reduce H+ produced by consuming acetic acid and butyric acid to hydrogen, and also shortened the photo-fermentation period. By scanning electron microscopy and X-ray diffraction analysis, the morphology and phase structure of TiO2/ACF after PFHP did not change significantly. This study laid the foundation for the reuse of TiO2 and its practical application in PFHP.

Enhancing fermentation yield for biohydrogen production using eco-friendly nickel and cobalt ferrite nanoparticles

AbstractIn recent years, the sustainable metal oxide nanoparticle synthesis using various plant leaf extracts as reducing agents has gained significant attention in the field of renewable energy research. This study explores the biohydrogen yield enhancement potential of two types of nanoparticles derived from olive leaves. Synthesized nanoparticles are characterized by SEM, EDX, and FTIR spectrum analyses; these nanoparticles exhibit mean dimensions of 385 nm and 292 nm. Fermentation experiments were conducted using varying ratios of the two types of nanoparticles ranging from 50 to 500 mg/L to determine the impact of these nanoparticles on bio-hydrogen yield. Remarkably, with the usage of 200 mg/L of nickel ferrite nanoparticles boosted biohydrogen yield by 47%, while 200 mg/L of cobalt ferrite nanoparticles increased it by 41%. These findings highlight the potential of sustainably synthesized nanoparticles, especially those from olive leaves, as catalysts for enhancing biohydrogen production in dark fermentation processes. This research offers promising insights into eco-friendly bioenergy generation.

Enhancement of biohydrogen production and low coke formation by applying Ni/ZrxCe1-xO2 catalyst in steam gasification of spent coffee ground in monolithic reactor

To produce H2-rich gas, steam gasification of spent coffee grounds is performed in this study by applying ZrxCe1-xO2 supports (x = 0.2, 0.6) in a monolithic reactor. Compared with conventional Ni/γ-Al2O3, Ni/ZrxCe1-xO2 catalysts offer higher gas yields and H2 generation. In particular, Ni/Zr0.6Ce0.4O2 exhibits the highest gas yield (86.32 wt%), H2 selectivity (56 vol%), and low coke formation owing to its favorable Ni dispersion, high oxygen mobility, and completely homogeneous nanometer-sized morphology. A significant increase in gas yield was observed in the monolith reactor (M@Ni/Zr0.6Ce0.4O2) compared to the fixed bed reactor (F@Ni/Zr0.6Ce0.4O2), from 74.85 wt% to 86.32 wt%. H2 selectivity (56.87 vol%) is also increased, which can be associated with the unique properties of the monolithic reactor. This phenomenon is attributed to the increase in residence time and the amount of mass and heat transfer between the catalyst and vapor, which favorably direct the selectivity toward H2. The use of metal-oxide composite supports, especially Ni/Zr0.6Ce0.4O2 catalyst, effectively alleviated coke formation (0.7 wt%), whereas the amount of coke produced by Ni/γ-Al2O3 (2.4 wt%) was more than three times higher. Overall, exploiting the remarkable benefits of ZrxCe1-xO2 (x = 0.2, 0.6) composite oxides as supports for Ni catalyst and monolithic reactors would have an emerging outlook for H2 generation via gasification technology.

Biohydrogen production from co-digestion of sugarcane vinasse and bagasse using anaerobic dark fermentation

Dark co-fermentation of agricultural waste for biohydrogen production is a promising avenue for sustainable renewable energy. This study investigates biohydrogen production from sugarcane-based substrates, specifically vinasse and bagasse. Sugarcane bagasse is hydrolyzed in acidified vinasse at various ratios (1:0, 1:15, 1:30, 1:45, and 0:1 dry weight (g)/liquid (mL)). The 1:15 ratio yields the highest hydrolyzed sugar concentration at 10.03 g sugar/L. Additionally, the optimal conditions for biohydrogen production are identified, with the 1:15 ratio demonstrating the maximum cumulative biohydrogen volume of 1462 NmL and a peak volumetric biohydrogen production rate (VHPRm) of 110.79 NmL H2/Lr.h. The pH optimum for biohydrogen production is determined to be 6.5, and production ceases below an initial pH of 4.5. Furthermore, under thermophilic conditions, the 1:15 ratio exhibits a VHPRm of 81.25 NmL H2/Lr.h and a hydrogen content (%H2) of 44.7 %. Overall, this study underscores the potential of co-digesting bagasse and vinasse to enhance biohydrogen production.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Comparative Analysis of Additives for Enhanced Biohydrogen Production via Dark Fermentation

Comparative Analysis of Additives for Enhanced Biohydrogen Production via Dark Fermentation

Enhancing biohydrogen production by peanut shell carrier assisted fermentation at different hydraulic retention time

The microbial immobilization and suitable hydraulic retention time (HRT) are conducive to maintain the biomass for further prompting the hydrogen production efficiency. In this study, peanut shell was employed as carriers to investigate the hydrogen production enhancement and the promotion mechanism at different HRTs with plastic pellet carrier as control. Both of the over-acid phenomenon and the operational stability decreasing occurred when HRT was shorter or longer than 6 h (HRT = 2 h, 4 h, or 8 h) in this study. When peanut shell was used as carrier, the maximum H2 yield of 1.7 L and H2 production rate of 0.58 L/L/d were obtained at HRT of 6 h, which were nearly 20 % and 15 % higher than that of plastic pellet as carriers, respectively. The hydrogen production system with peanut shell as carriers had better hydrogen production capacity and immobilized effect, since peanut shell provided a supplementary substrate for the growth and metabolism of hydrogen producing bacteria, and buffered the acidity of the system. This study provided an application for immobilization technology and the industrialization of bio-hydrogen production.

New insights into enhancement of bio-hydrogen production through encapsulated microalgae with alginate under visible light irradiation

The production of green hydrogen is a promising alternative to fossil fuels. The current study focuses on the design of microalgae as a catalyst in bioelectrochemical systems for the generation of biohydrogen. Furthermore, the abovementioned target could be achieved by optimizing different parameters, including strains of microalgae, different optical filters, and their shapes. Synechocystis sp. PAK13 (Ba9), Micractinium sp. YACCYB33 (R4), and Desmodesmus intermedius (Sh42) were used and designed as free cells and immobilized microalgae for evaluating their performance for hydrogen production. Alginate was applied for immobilization not only for protecting the immobilized microalgae from stress but also for inhibiting the agglomeration of microalgae and improving stability. The amount of studied immobilized microalgae was 0.01 g/5 ml algae-dissolved in 10 ml alginate gel at 28 °C, 12 h of light (light intensity 30.4 μmol m−2 s−1), and 12 h of darkness with continual aeration (air bump in every strain flask) at pH = 7.2 ± 0.2 in 0.05 %wuxal buffer which has 3.7 ionic strength. Different modalities, including FTIR, UV, and SEM, were performed for the description of selected microalgae. The surface morphology of Ba9 with alginate composite (immobilized Ba9) appeared as a stacked layer with high homogeneity, which facilitates hydrogen production from water. The conversion efficiencies of the immobilized microalgae were evaluated by incident photon-to-current efficiency (IPCE). Under optical filters, the optimum IPCE value was ∼ 7 % at 460 nm for immobilized Ba9. Also, its number of hydrogen moles was calculated to be 16.03 mmol h−1 cm−2 under optical filters. The electrochemical stability of immobilized Ba9 was evaluated through repetitive 100 cycles as a short-term stability test, and the curve of chrono-amperometry after 30 min in 0.05 %wuxal at a constant potential of 0.9 V for 30 min of all studied samples confirmed the high stability of all sample and the immobilized Ba9 has superior activity than others.

Enhanced biohydrogen production from high loads of unpretreated cattle manure by cellulolytic bacterium Caldicellulosiruptor bescii at 75 °C

Caldicellulosiruptor bescii is the most thermophilic cellulolytic bacterium capable of fermenting crystalline cellulose identified to date, and it also has a superior ability to degrade plant biomass without any pretreatment. This study is the first to assess the potential of utilizing unpretreated cattle manure (UCM) as a feedstock for hydrogen (H2) production by C. bescii at a concentration range between 2.5–50 g volatile solids (VS)/L. At 50 g VS/L UCM concentrations, H2 production ceased due to inhibition of C. bescii. To alleviate the impacts of inhibition, two strategies were adopted: (i) reduction of H2 build-up in the reactor headspace via gas sparging and (ii) adaptation of C. bescii to UCM via adaptive laboratory evolution (ALE). The former increased H2 yield by 47% compared to the control reactors, where no sparging was applied. The latter increased H2 yield by 142% compared to the control reactors inoculated by the wild type C. bescii. The UCM-adapted C. bescii demonstrated a remarkable H2 yield of 161.3 ± 1.6 mL H2/g VSadded at 15 g VS/L. This yield represents a twofold increase compared to the maximum H2 yield reported in the literature amongst fermentation studies utilizing manure as feed. At 15 g VS/L, around 73% of UCM was solubilized, and the carbon balance indicated that most of the effluent carbon was in the sugar- and acid-form. The remarkable ability of C. bescii to produce H2 from UCM under non-sterile conditions presents a significant potential for sustainable biohydrogen production from renewable feedstocks.

Enhancing bio-hydrogen and bio-methane production of concentrated latex wastewater (CLW) by Co-digesting with palm oil mill effluent (POME): Batch and continuous performance test and ADM-1 modeling

This study aimed at investigating the biohydrogen and biomethane potential of co-digestion from palm oil mill effluent (POME) and concentrated latex wastewater (CLW) in a two-stage anaerobic digestion (AD) process under thermophilic (55 ± 3 °C) and at an ambient temperature (30 ± 3 °C) conditions, respectively. The batch experiments of POME:CLW mixing ratios of 100:0, 70:30, 50:50, 30:70, and 0:100 was investigated with the initial loadings at 10 g-VS/L. The highest hydrogen yield of 115.57 mLH2/g-VS was obtained from the POME: CLW mixing ratio of 100:0 with 29.0 of C/N ratio. While, the highest subsequent methane production yield of 558.01 mLCH4/g-VS was achieved from hydrogen effluent from POME:CLW mixing ratio of 70:30 0 with 21.8 of C/N ratio. This mixing ratio revealed the highest synergisms of about 9.21% and received maximum total energy of 19.70 kJ/g-VS. Additionally, continuous hydrogen and methane production were subsequently performed in a series of continuous stirred tank reactor (CSTR) and up-flow anaerobic sludge blanket reactor (UASB) to treat the co-substate. The results indicated that the highest hydrogen yield of POME:CLW mixing ratio at 70:30 of 95.45 mL-H2/g-VS was generated at 7-day HRT, while methane production was obtained from HRT 15 days with a yield of 204.52 mL-CH4/g-VS. Thus, the study indicated that biogas production yield of CLW could be enhanced by co-digesting with POME. In addition, the two-stage AD model under anaerobic digestion model no. 1 (ADM-1) framework was established, 9.10% and 2.43% of error fitting of hydrogen and methane gas between model simulation data and experimental data were found. Hence, this research work presents a novel approach for optimization and feasibility for co-digestion of POME with CLW to generate mixed gaseous biofuel potentially.

Influence of iron and magnesium on kinetic and mechanism of biohydrogen production from high-strength wastewater

In this study, iron/iron-magnesium (Fe/Fe–Mg) additives were prepared through the impregnation of granular activated carbon (GAC) with iron and iron-magnesium (GFM) to enhance biohydrogen production. The microscope observation and chemical analysis revealed that the GAC matrixes were well infused with Fe/Fe-Mg, while the X-ray diffraction analysis revealed the species of metal formed on the GAC as Fe3+ and MgH2. The synergistic effect of Fe and Mg in GFM allowed it for a shorter delay time and higher hydrogen production rate than other additives, indicating their possible use in stimulating the fast release of hydrogen in anaerobic digestion. The co-metabolites analysis revealed that additives ensured biohydrogen production through the different pathways. The plausible mechanisms were through hydrogenases ensured by Fe3+ and hydrolysis by MgH2. GFM gave the best organic matter and nutrient removal efficiency to outperform other additives, suggesting its ability for biohydrogen synthesis and simultaneous wastewater treatment.

Optimizing biohydrogen production yields by employing locally isolated thermophilic bacteria from hot springs

A climate-neutral economy is anticipated to rely heavily on hydrogen because it enables emission-free transportation, heating, and manufacturing operations. Biohydrogen can be produced from various kinds of biological waste making the interest high. However, the yield and efficiency of the processes are still challenging. This study applied Box-Behnken statistical experimental design to investigate the influence of temperature (oC), pH, and CO volume (mL) together with the amount of Fe+2, Zn+2, and Ni+2 to enhance biohydrogen production yields from thermophilic cultures, both mixed and pure cultures isolated from hot springs in Izmir, Türkiye. The maximum H2 yields were reported as 0.13 mmolH2/mmolCO for mixed cultures, and the pure culture reached 2.5 fold higher yield (0.44 mmolH2/mmolCO). Bench-scale bioreactor with a custom-design micro sparger was successfully run for 7 days (highest 0.25 mmolH2/mmol CO). This is the first report in the literature with local isolates to demonstrate the optimization of H2 yields with a comparative approach, and scale-up in a 2 L bench scale bioreactor. The viability of using novel thermophilic isolates as biohydrogen producers was successfully proven.