bio-H2 Production Research Articles

Application of green synthetic iron oxide nanocatalyst in biohydrogen production from domestic wastewater.

Hydrogen (H2) is considered to be an energy carrier with high yields in future. Using green nanoparticles as the catalyst to produce H2 from organic wastes is an environmentally friendly and cost-effective approach. This study specifically addresses H2 production biologically from domestic wastewater using dark fermentation process with green nanoparticles in anaerobic condition. In this investigation, acid, alkaline, and heat pre-treated wastewater were utilized as the feedstock and identified that the alkaline pre-treated wastewater was an efficient feedstock for H2 production. The reaction conditions such as pH, time, and nanocatalyst dosage were optimized for the enhancement of H2 production. In addition, the green iron oxide nanoparticles (Fe3O4-NPs) were prepared by sustainable way using Murraya koenigii (curry leaves) with FeSO4 and characterized it using Fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible spectrophotometer (UV-vis), power X-ray diffraction (pXRD), and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM/EDX), further tested for their 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging performance via in vitro antioxidant activities. This study demonstrates that a maximum production of bio-H2 was obtained (72.8mL/L) with 20mg of nanocatalyst load at alkaline condition (pH 9). The correlation coefficient value was highly significant for biohydrogen production with increasing pH (r = 0.9026) and catalyst dosage (r = 0.9962). The characterization studies confirmed the existence of iron oxide nanoparticles in the green synthesized nanoparticles. The in vitro antioxidant assay showed that Fe3O4-NPs effectively reduces the DPPH radical by significantly releasing H-atoms, thereby confirming that the plant extract functions as reducing/oxidizing agents. The high yield under the dark fermentation method has clearly been attributed to the enhancement in bio-H2 production by using the green Fe3O4-NPs based on M. koenigii.

Hydrogen production from energetic poplar and waste sludge by electrohydrogenesis using membraneless microbial electrolysis cells

Membraneless microbial electrolysis cells (MECs) are potentially considered to produce biohydrogen (bioH2) in a green manner and simultaneously minimize agricultural and wastewater facility wastes. However, effective, sustainable, and cost-effective system configuration and improvement of operating variables, working at ambient conditions, are needed to make the MEC a sustainable process. Therefore, this study investigates the bioH2 production from poplar leaves and anaerobic sludge mixture by incorporating nanomaterials comprising Al2O3, MgO, and Fe2O3 metal oxides at various dosages. Moreover, the effects of applied cell voltage (0.5–1.5 V) and inoculum amount (20–40 mL) on bioH2 production and organic matter removal performance are evaluated. The maximum bioH2 production value is 417 mL at an applied voltage of 1.5 V with a chemical oxygen demand (COD) removal efficiency of 37.6 % under operating times of 5 min using 40 ml of inoculum. The bioH2 production of the MEC system is reduced with the decrease in inoculum amount. The highest bioH2 production of 828 mL is obtained at improved conditions in the presence of 1 g of Fe2O3 metal oxide. Overall, this study provides the potentiality of simultaneous waste minimization and bioH2 production under ambient conditions that highlight the waste-to-energy pathway for membraneless and green bioelectrochemical process.

Potential and characteristics of bio-H2 production from the garden waste of ornamental plant Malus prunifolia fruit: Comparison between ethanol-type and butyrate-type fermentations

Edible fruit waste has been studied and considered to be the ideal substrates for low-cost bio-H2 production. However, the garden waste of ornamental plant fruits, which has similar components to edible fruit, is rarely studied. In this study, the potential and characteristics of fermentative bio-H2 production from the campus ornamental plant fruit of Malus prunifolia (MP) in both ethanol- and butyrate-types were investigated. Ethanoligenens harbinense B49 and Clostridium neuense G1 were employed as the inoculums for the ethanol- and butyrate-type fermentations, respectively. The results indicated that the fruit of MP is rich in fructose and sucrose, both of strains B49 and G1 could grow well with MP powder as the carbon source for fermentation. However, a higher H2 yield of 1.77 mol-H2/mol-hexose was obtained in the butyrate-type fermentation with strain G1, which was 1.4 times of that with strain B49. C. neuense G1 was more suitable for the H2 fermentation with MP as substrate, and a H2 yield of 0.55 mol-H2/mol-hexose by strain B49 was observed in the test of buffer system exchanging from ethanol-type to the butyrate-type. In summary, garden wastes such as fruits of ornamental plants should be given more attention as low-cost fermentation substrate candidates for bio-H2 production.

Comparison of single-vs dual-chamber microbial electrolysis cells performance for biohydrogen production

Abstract In this study, fed batch single- and dual-chamber microbial electrolysis cells (MECs) were operated to compare differences in the electrohydrogenesis performance at 0.8 V applied voltage and hydraulic retention time (HRT) of 48 h. Results showed that current intensity, current density, COD removal efficiency, and H2 production rate were higher in the dual-chamber MEC than in the single-chamber configuration. Electromethanogenesis and homoacetogenesis scavenged the H2 from electrohydrogenesis thus reducing H2 generation, as demonstrated by cathodic efficiencies less than 10%. It appears that homoacetogenesis overshadowed H2 production in the dual-chamber MEC, as reflected by a coulombic efficiency greater than 100%, likely induced by a high H2 partial pressure. These results portray MECs as an energy-efficient scheme for bioH2 production in addition to removal of pollutants (COD). This study presented valuable information when deciding on suitable reactors and optimizing reactor design.

Biohydrogen production from wastewater: Production technologies, environmental and economic aspects

Biohydrogen (bio-H2) production from wastewater can be a sustainable and innovative approach that uses biotechnological processes to convert organic waste into hydrogen, a clean energy source. Technologies such as dark fermentation, photo-fermentation, microbial electrolysis cells, and biophotolysis are widely utilized, with emerging technologies like anaerobic membrane bioreactors and microalgae processes also gaining prominence. Integration between these systems aims to enhance process efficiency. This study reviews the hydrogen production from wastewater, analyzing its potential as a source for bio-H2 production and evaluating environmental and economic issues. The performance of various processes in bio-H2 production from different wastewater sources is discussed, focusing on challenges, opportunities, and trends. Wastewater, including cassava (225.2 mL H2/gVS) and corn (265 mL H2/gVS) processing, dairy (54.5 mL H2/gVS), domestic (154–180 mL H2/gVS), fruit (104.9 mL H2/gVS) and starchy (153–200 mL H2/gVS), show promising potential for bio-H2 production. Economically, indirect biophotolysis is the most cost-effective technology per kg of bio-H2 produced, followed by microbial electrolysis cells, photo-fermentation, dark fermentation, and direct biophotolysis. Furthermore, from an environmental and sustainability perspective, bio-H2 production presents a strategic tool for global decarbonization efforts, reducing CO2 emissions and facilitating transformation towards clean energy solutions. Bio-H2 generation during wastewater treatment holds potential by concurrently producing clean fuel and enabling safe disposal of wastewater. Continued research and development efforts are essential to overcoming current challenges. Achieving this will require targeted investments, policy support, and technological innovations to make biohydrogen a viable and sustainable energy solution.

Bioaugmentation with Clostridium pasteurianum for high-yield continuous bio-hydrogen production in a dynamic membrane bioreactor

This study demonstrated the feasibility of achieving high-yield continuous bio-H2 production through bioaugmentation with Clostridium pasteurianum. C. pasteurianum was introduced during the initial granulation stage in a dynamic membrane bioreactor (DMBR) inoculated with heat-treated digester sludge. Following bioaugmentation, the average hydrogen yield (HY) reached 3.07 ± 0.13 mol H2/mol hexoseconsumed, with a hydraulic retention time (HRT) of 2 h. This high-yield and high-rate hydrogen production performance was achieved alongside a high concentration of suspended and granular biomass as 21.02 ± 1.01 g VSS/L. The DM played a crucial role in prolonging the solid retention time of the suspended biomass to 12.15 h. C. pasteurianum emerged as the predominant species, constituting 92.3 % of the total bacterial population. This dominance significantly contributed to achieving the high HY, reaching 77 % of the theoretical maximum HY (4 mol H2/mol hexoseconsumed) by shifting the metabolic pathway from non-H2 production pathways to hydrogen-producing acetate. However, during further continuous operation, the hydrogen production pathway was gradually shifted towards the butyrate pathway, resulting in a decreased HY of 2.26 mol H2/mol hexoseconsumed along with the decreased population of C. pasteurianum. Additional bioaugmentation of C. pasteurianum during the maturational granulation stage did not impact the metabolic pathways or microbiome significantly, as the added strain was supposed to be washed out. This study provides insights into achieving high-yield continuous bio-H2 production through bioaugmentation with C. pasteurianum during the initial granulation stage. Further studies are necessary to sustain C. pasteurianum-dominated high HY with the acetate pathway during prolonged operation.

High-calorific biohydrogen production under high pressure: Ca2+ addition, theoretical prediction, and continuous operation

The removal of CO2 (called “upgrading”) is essential in bio-H2 production processes including dark fermentation (DF), which is energy-intensive and adds economic burden. Here, it was attempted to generate high calorific bio-H2 (high H2-content) directly from the fermenter under high pressure in the presence of Ca2+. High pressure was automatically generated by holding the produced gas at a certain limit. Batch test results showed that Ca2+ addition was helpful to keep the pH by precipitating with dissolved CO2, resulting in the gradual increase of H2 content to 93 % at 17.5 bar (vs. 77 % at 11.2 bar in the control). Then, the theoretical approaches, based on balance and equilibrium equations, were made to investigate the effects of Ca2+ addition at different dosages (0–1 g CaCl2/L) under different pressures during continuous operation. It was found that the H2 content of 90 % at the permissible pressure (≤10 bar) can be only achieved at 1 g CaCl2/L addition. Lastly, continuous operation of DF was conducted in an up-flow anaerobic sludge blanket reactor fed with glucose (10 g COD/L) with 1 g CaCl2/L addition. The H2 content in bio-H2 gradually increased with the pressure increase, from 49.5 % at 1 bar to 84.9 % at 9 bar. No distinct changes in morphological and physico-chemical properties were observed in the granules exposed at different pressures. Although there was a drop in H2 production due to the increase in dissolved H2 portion and metabolic shift at high pressure, the decrease in upgrading cost made high-pressure DF economically feasible.

Evaluating the feasibility of carbon fiber brush for improving biohydrogen production in acidogenic dark fermentation

Dark fermentation is a promising technology for biohydrogen (bioH2) production. Augmenting the dark fermentation process with conductive materials has drawn significant research interests improving the bioH2 yield. This study evaluated the feasibility of using carbon fiber brush (CB) as an additive to increase the efficiency of mesophilic dark fermentation using glucose as substrate and heat-shock pretreated anaerobic sludge as inoculum. Effect of electrode packing density on bioH2 yield, soluble metabolite product formation, and substrate degradation was investigated. Maximal bioH2 yield of 334.6 mL/g-glucose was obtained using medium-sized CB with 72.5% higher than the control (194 mL/g-glucose) and butyrate-type fermentation was the dominant metabolic pathway. Further, enhanced concentrations of proteins and polysaccharides on CB biofilm suggested the critical role of extracellular polymeric substances in microbial electron transfer. This was supported via electron transport activity in CB biofilm. Finally, microbial community analysis revealed a selective enrichment of the potentially electroactive Clostridium_sensu_stricto_1 on the CB (56.6%) compared to 40.9% abundance in control. Insights gained from this study demonstrated that CB aided dark fermentation can be an economical and environmentally benign strategy for improving bioH2 yield.

Efficient bio-hydrogen production by dark co-fermentation of food-rich municipal solid waste and urban pruning wastes of pine, cypress, and berry

Bio-hydrogen (bio-H2) production from dark co-fermentation of pruning wastes, from pine, cypress, and berry trees, and food-rich municipal solid waste (FMSW) was evaluated as a promising way to combine waste management and renewable energy production. The inoculum was initially optimized by heat-shock pretreatment at 65, 75, 85, and 95 ℃ for 15, 30, and 45 min to maximize bio-H2 yields. The optimum pretreatment conditions (75 °C for 45 min) were applied for dark co-fermentation analyses. The highest bio-H2 production yield of 84 ± 6 mL/g VS was obtained by dark co-fermentation of 20 g/L pruning wastes and FMSW under initially neutral conditions. The fermentation of individual substrates led to 40.9, 0.2, 0.1, and 0.2 mL/g VS from FMSW, pine, berry, and cypress, respectively, 85% lower than that obtained through dark co-fermentation. Therefore, co-processing of a “stiff substrate”, lignocellulose, with a “loose part”, starchy FMSW, leads to a higher bio-H2 yield. Dark co-fermentation led to the bio-conversion of more than 76% of starch, 31% of glucan, and 41% of hemicellulose, besides 41% lignin removal. The modified Gompertz model (MGM) and Logistic model (MLM) were well-fitted on kinetic data (with R2 values ≥0.98), and the kinetic parameters were calculated.

Microbial electrolysis cells designed for BioH2 production: Potential improvements with carbon dioxide and nitrogen

The long-term feasibility and efficiency of microbial electrolysis cells (MECs) depend primarily upon several crucial parameters. However, they show promise for sustainably producing biohydrogen (bioH2). This study investigates a newly designed biohydrogen reactor by studying the effects of several variables on the production of bioH2, including applied voltage, electrode material, and gas sparging with CO2 and N2. The study aims to optimize the system for improved efficiency and production performance. The results of this study show that both electrode type and applied voltage affect bioH2 production where choosing the right inoculum has a significant impact on the catalytic efficiency. The aluminum electrodes significantly help produce the most bioH2 production at 2.0 V with 884 mL/L in 11.25 min, while emphasizing the significance of material characteristics. The reduced partial pressure from CO2 sparging, up to a point, improves bioH2 generation. The N2 gas sparging further demonstrates an increased efficiency where the ideal flow rates are dependent on the size of the MEC system. The highest bioH2 production rates for CO2 and N2 sparging are measured as 821 mL/L and 813 mL/L in 4.25 min and 11.50 min at a volume of 400 mL/min and 300 mL/min, respectively. The results of this study further advance our knowledge of and ability to optimize MEC systems for long-term, sustainable bioH2 production.

Development of a unique integrated bioreactor for simultaneous desalination and bioenergy and biohydrogen production

In the wastewater treatment challenge, it is really essential to develop integrated systems in reducing greenhouse gases, producing green energy and achieving sustainable development. In this regard, an integrated electro-biomembrane reactor was developed and performed in this study for simultaneous biohydrogen (bioH2) production from energetic poplar leaves using dark fermentation (DF) process, conventional H2 production, bioenergy production in the DF process, and saline water desalination in a single system. The results of this study showed that pH was the main controlling parameter in bioH2 production, and the superior production of 40.2 mL/g-biomass was obtained at a pH of 5.5. The maximum current and power density values were 2861.7 mW/m2 and 2819.4 mA/m2 at pH 5.5 under improved conditions. Furthermore, the maximum conventional H2 production was found to be 1341.6 mL using 2 M of KOH solution. Overall, the results further proved that the proposed integrated system can be a sustainable and promising process for industrial applications, considering its high desalination, energy production, and conventional and biological H2 production efficiencies.

Design of a microbial photoheterotrophic consortia for biohydrogen production under nongrowing conditions: Insight into microbial associations

Physiological and metabolic behavior of photoheterotrophic mixed cultures (PHMCs), monocultures of Rhodopseudomonas palustris, Clostridium pasteurianum and Syntrophomonas wolfei and a designed microbial consortium (DMC), consisting of these microorganisms that emulates natural consortia were studied. Growing and photoheterotrophic nongrowing conditions to simulate the use of dark fermentative effluents were used. Under growing conditions, C. pasteurianum showed higher bioH2 production (8.5 mmol) and molar bioH2 yield (21 mmoLH2/gCOD), while R. palustris did not produce this gas. Under nongrowing conditions, DMC reached the highest bioH2 production (10 mmol) and bioH2 yield (78.6 mmoLH2/gCOD) together with Poly-hydroxy-alkanoates (PHA), followed for PHMC-C2 (7.5; 59.6) and the monocultures. Higher bioH2 and PHA production of the PHMC and the DMC suggest that these conditions promote the use of nonconventional energy pathways, and interactions among microbial populations that allow for the survival and sustain bioH2 production. This study forms the basis for more in-depth studies of the metabolic behavior of photosynthetic natural and designed cultures.

Production of biological hydrogen from Quinoa residue using dark fermentation and estimation of its microbial diversity

Although they are one of the world's environmental problems, agricultural wastes or residues are carbohydrate-rich and low-cost, so they are used as raw materials for the manufacture of biohydrogen (bio-H2). Among biological hydrogen manufacture methods, the dark fermentation method is suitable for processing waste or residues. In this regard, no study has been found in the literature on determining the potential of biological hydrogen manufacture from quinoa residue by the dark fermentation method. This work was carried out in a dark room at 36 ± 1 °C under different operating conditions in anaerobic batch bio-reactors fed with thermally pretreated anaerobic mixed bacteria + raw quinoa or quinoa extract liquid + nutrients. In the study, gas analyses were performed and biohydrogen production was detected in all the bio-reactors. Besides, taxonomic content analyses and organic acid analyses were executed. Maximum bio-H2 production was found as follows: at pH 4.5, 14,543.10−4 mL in the bio-reactor fed with 1.00 g quinoa/L and 1880.10−4 mL in the bio-reactor fed with 0.50 g quinoa extract/L, and at pH 4.0, 61,537.10−4 mL in the bio-reactor fed with 1.00 g quinoa/L and 1511.10−4 mL in the bio-reactor fed with 0.75 g quinoa extract/L. In the bio-reactors fed with raw quinoa residue, Clostridium butyricum and Hathewaya histolytica were detected as the most dominant bacteria at pH 4.5 and 4.0, respectively, whereas in the bio-reactors fed with quinoa extract liquid, Fonticella tunisiensis were detected as the most dominant bacteria at both pH 4.5 and pH 4.0.

A novel two-stage immobilized bioreactor for biohydrogen production using a partial microalgal-bacterial (Chlorella vulgaris and wastewater activated sludge) co-culture

Biohydrogen (bioH2) has the potential to be a sustainable and carbon–neutral source of bioenergy, as it does not produce any greenhouse gas emissions. Various strategies and techniques such as algal-bacterial co-culturing and bioreactor configurations have been applied to improve bioH2 yield and regulate process inhibitors (low pH and high organic acids yield). Therefore, in current study, the co-culturing of Chlorella vulgaris (C. vulgaris) and domestic wastewater activated sludge (WWAS) was exploited in an innovative way by immobilization in two separate bioreactors. The C. vulgaris and WWAS were immobilized in sodium alginate and Z-medium was utilized as a source of nutrient supplemented with 10 g/L of glucose as external carbon source. The maximum bioH2 production of 9.8 and 222.7 mL/L was obtained in microalgal and bacterial chambers, respectively. Acetic acid accumulation was reduced to below 150 mg/L which is 90 % less compared to previous studies and subsequently the pH raised ranging from 7.0 to 10.0 during six days of incubation period. The results suggest that immobilized microalgae and WWAS in partial co-culture tend to reduce process inhibitors by increasing pH via microalgal photosynthesis and bioH2 production via sugar fermentation, respectively. The strategy to split microalgal-bacterial co-culture into separate chambers provides the ability to work microalgae and bacteria separately but in a symbiotic manner.

Combining pretreatments and co-fermentation as successful approach to improve biohydrogen production from dairy cow manure

The present paper is focused on enhancing the production of biohydrogen (bioH2) from dairy cow manure (DCM) through dark fermentation (DF). Two enhancement production strategies have been tested: i) the combination of H2O2 with sonification as pretreatment and ii) the co-fermentation with cheese whey as co-substrate. Concerning the pretreatment, the best combination was investigated according to the response surface methodology (RSM) by varying H2O2 dosage between 0.0015 and 0.06 g/gTS and ultrasonic specific energy input (USEI) between 35.48 and 1419.36 J/gTS. The increase of carbohydrates concentration was used as target parameter. Results showed that the combination of 0.06 g/gTS of H2O2 with 1419.36 J/gTS of USEI maximized the concentration of carbohydrates. The optimized conditions were used to pretreat the substrate prior conducting DF tests. The use of pretreatment resulted in obtaining a cumulative bioH2 volume of 51.25 mL/L and enhanced the bioH2 production by 125% compared to the control test conducted using raw DCM. Moreover, the second strategy, i.e. co-fermentation with cheese whey (20% v/v) as co-substrate ended up to enhancing the DF performance as the bioH2 production reached a value of 334.90 mL/L with an increase of 1372% compared to the control DF test. To further improve the process, dark fermentation effluents (DFEs) were valorized via photo fermentation (PF), obtaining an additional hydrogen production aliquot.

Evaluation of short-circuited electrodes in combination with dark fermentation for promoting biohydrogen production process

Short-circuited electrodes, in combination with dark fermentation, were evaluated in a biohydrogen production process. The system is based on an innovative design of a non-compartmented electromicrobial bioreactor with a conductive tubular membrane as cathode and a graphite felt as anode. In particular, the electrode specialization occurred when the bioreactor was inoculated with manure as the whole medium and when a vacuum was applied in the tubular membrane, for allowing continuous extraction of gaseous species (H2, CH4, CO2) from the bioreactor. This specialization of the electrodes as anode and cathode was further confirmed by microbial ecology analysis of biofilms and by cyclic voltammetry measurements. In these experimental conditions, the potential of the electrochemical system (short-circuited electrodes) reached values as low as −320 mV vs. SHE, associated with a significant bioH2 production. Moreover, a higher bioH2 production occurred and a potential of the electrochemical system as low as −429 mV vs SHE was temporarily observed, when additional heat treatments of the whole manure were applied in order to remove methanogen microorganisms (i.e., hydrogen consumers). In the bioreactor, the higher production of bioH2 would be promoted by electrofermentation from the current flow observed between short-circuited anode and cathode.

Biological H2(g) Production and Modelling with Computational Fluid Dynamics (CFD)

In this study, bio-hydrogen gas [bio-H2(g)] production and modeling with a three-phase computational fluid dynamics (CFD) model, heat and mass transfer of bio-hydrogen production, reaction kinetics, and fluid dynamics; It was investigated by dark fermentation process in an anaerobic continuous plug flow reactor (ACPFR). The three-phase CFD model was used to determine the bio-H2(g) production in an ACPFR. The effect of different operating parameters, increasing hydrolic retention times (HRTs) (1, 2, 4, 8, and 12 days), different pH values (4.0, 5.0, 6.0, 7.0, and 8.0), and increasing feed rate as organic loading rates (OLRs) (0.5, 1.0, 2.0, 4.0, 8.0 and 10.0 g COD/l.d) on the bio-H2(g) production rates were operated in municipal sludge wastes (MSW) with Thermoanaerobacterium thermosaccharolyticum SP-H2 methane bacteria during dark fermentation for bio-H2(g) production. The effect of HRT, pH, and feed rate on the bioH2(g) efficiencies and H2(g) production rates were examined in the simulation stage. Production of volatile fatty acids (VFAs) namely, acetic acids, butyric acids, and propionic acids were important points influencing the bio-H2(g) production yields. The artificial neural network (ANN) model substrate inhibition on bio-H2(g) production to the methane (CH4) bacteria was also investigated. The reaction kinetics model used Thermotoga neapolitana microorganisms with the Andrews model of substrate inhibition. Furthermore, the ANN model was well-fitted to the experimental data to simulate the bio-H2(g) production from chemical oxygen demand (COD).

Potential of Bacillus subtilis as oxygen-removal agent for biohydrogen production by Clostridium acetobutylicum

Molasses are a source of fermentable sugars, the substrate utilized for this study, as it is a subproduct from sugarcane processing for sucrose recovery. These sugars can be metabolized for bioH2 production by bacterial strains, particularly strict-anaerobe Clostridium acetobutylicum (Clac) and its media preparation requires chemical of O2 removal and N2 flushing to promote anaerobiosis. Bacillus subtilis (Bsub) can deplete O2 from culture media and promotes anaerobiosis, which is an aspect that would benefit from further research. Clac was grown in an anaerobic medium and produced 269 mL of hydrogen at 72 h. By co-culturing Clac and Bsub, 502 mL of bioH2 were produced in a cysteine/N2 flushed medium, while 203 mL of bioH2 at 72 h were produced by aerobic co-culture. Additionally, in an untreated/aerobic conventional growth culture for Clac, no production of H2 was detected, reinforcing the utility of Bsub for anaerobiosis generation.

Using custom-built primers and nanopore sequencing to evaluate CO-utilizer bacterial and archaeal populations linked to bioH2 production

The microbial community composition of five distinct thermophilic hot springs was effectively described in this work, using broad-coverage nanopore sequencing (ONT MinION sequencer). By examining environmental samples from the same source, but from locations with different temperatures, bioinformatic analysis revealed dramatic changes in microbial diversity and archaeal abundance. More specifically, no archaeal presence was reported with universal bacterial primers, whereas a significant archaea presence and also a wider variety of bacterial species were reported. These results revealed the significance of primer preference for microbiomes in extreme environments. Bioinformatic analysis was performed by aligning the reads to 16S microbial databases for identification using three different alignment methods, Epi2Me (Fastq 16S workflow), Kraken, and an in-house BLAST tool, including comparison at the genus and species levels. As a result, this approach to data analysis had a significant impact on the genera identified, and thus, it is recommended that use of multiple analysis tools to support findings on taxonomic identification using the 16S region until more precise bioinformatics tools become available. This study presents the first compilation of the ONT-based inventory of the hydrogen producers in the designated hot springs in Türkiye.

Biohythane Production from Domestic Wastewater Sludge and Cow Dung Mixture Using Two-Step Anaerobic Fermentation Process

The current study explored bioenergy, particularly biohythane (a combination of biohydrogen (bioH2) and biomethane (bioCH4)), production from cow dung and untreated domestic wastewater sludge to valorize the waste into a value-added product. The experimental study consisted of a two-step process: dark fermentation (DF) and anaerobic digestion (AD) with a range of processing conditions varying the temperature and pH (acidic, neutral, and basic). The study maintained thermophilic conditions (55 °C) for bioH2 production and mesophilic conditions (35 °C) for bioCH4 production. The highest yields of bioH2 and bioCH4 were obtained at a pH of 5.5 (108.04 mL H2/g VS) and a pH of 7.5 (768.54 mL CH4/g VS), respectively. Microorganisms, such as Lactobacillus brevis and Clostridium butyricum, in the wastewater sludge accelerated the conversion reaction resulting in the highest bioH2 yield for an acidic environment, while Clostridium and Bacilli enhanced bioCH4 yield in basic conditions. The maximum cumulative yield of biohythane was obtained under basic pH conditions (pH 7.5) through DF and AD, resulting in 811.12 mL/g VS and a higher volumetric energy density of 3.316 MJ/L as compared to other reaction conditions. The experimental data were modelled using a modified Gompertz’s model at a 95% confidence interval and showed the best-fitting data from experimental and simulation results for biohythane production. The regression coefficient R2 value was highly significant at 0.995 and 0.992 for bioH2 and bioCH4 with the change in pH during biohythane production. Thus, this study presented an effective pathway to utilize untreated domestic wastewater sludge as an inoculum, showcasing the potential of biohythane production and the generation of valuable metabolic end-products across a broad range of pH conditions.