Dark Fermentative Hydrogen Production Research Articles

Effects of Sugar Beet Pulp Pretreatment Methods on Hydrogen Production by Dark Fermentation

Methane and hydrogen generated from waste and biomass are renewable resources, which may successfully replace traditional fossil fuels. This paper investigates the enhancement effect of lignocellulosic biomass pretreatment on dark fermentative hydrogen production from sugar beet pulp (SBP). The results showed that sugar beet pulp after pretreatment contained significant amounts of unfermented sugars (mainly glucose, arabinose, galactose, and raffinose), and, therefore, represented an attractive substrate for methane and hydrogen production. The greatest methane yield (495 dm3 CH4/kg VS) was achieved from sugar beet pulp after alkaline pretreatment. High methane production of up to 445 dm3 CH4/kg VS was also obtained using acidic and enzymatic hydrolysis as a preliminary treatment of the pulp. All the pretreatment methods also resulted in the enhancement of hydrogen yield with the highest value of 229 dm3 H2/kg VS achieved using acid hydrolysis compared with 17 dm3 H2/kg VS for raw material subjected to digestion.

Enhancing dark fermentative hydrogen production from wheat straw through synergistic effects of active electric fields and enzymatic hydrolysis pretreatment

Hydrogen, a clean and sustainable energy source, faces challenges from energy-intensive pre-processing technologies. This study explores the synergistic enhancement of active electric fields on enzymolysis of wheat straw and hydrogen production through dark fermentation. The active electric field enzymolysis system improved the adsorption capacity of wheat straw to cellulase, increasing cellulase activity by 18.0 %, causing a 39.1 % increase in reducing sugar content. In the active fermentation system, Clostridium_sensu_stricto_1 activity was enhanced in the first stage, increasing hydrogenase activity by 23.0 %, prolonging the first hydrogen production peak. Elevated reducing sugars were observed in the second stage, with Prevotella_9 and Bacteroides becoming the dominant hydrogen-producing bacteria in the third stage, leading to a second hydrogen production peak. Overall, cumulative hydrogen production was enhanced by 50.9 % compared to the control. The synergistic pretreatment with an active electric field and cellulase provides a novel approach for efficiently utilizing wheat straw.

Sustainable duckweed biomass valorization for dark fermentative hydrogen production: Process evaluation under meso- and thermophilic operations

Aquatic plants are rich in biomass and contain good amounts of carbohydrates and proteins which are amenable for valorization to generate bioenergy. Duckweed is rich in proteins and carbohydrates that grow abundantly in contaminated waterbodies, which is now tested for as a substrate for biohydrogen production. Dark-fermentative hydrogen production was done by hydrothermal pretreatment of duckweed biomass under 15 lbs pressure, evaluated under three different operating temperatures that cover mesophilic (35 °C) and thermophilic conditions (50 and 55°C). The study resulted in the maximum hydrogen production rate of 0.97 mmol/day in batch culture (160 mL) from 55 °C that was found superior to 50 °C (0.84 mmol/day) and 35 °C (0.38 mmol/day) operations. From the 7 days of operation, under uncontrolled pH conditions, substrate (chemical oxygen demand, COD) utilization was 57.27 %, which is higher than in mesophilic conditions (50.67 %). Efficient hydrolysate conversion was noticed with thermophilic conditions, especially under operation at 55 °C (416 mL H2/g of biomass) than mesophilic conditions (144 mL H2/g of biomass) suggesting that duckweed can be dependable biomass for hydrogen production, and the dark-fermentation can be an appropriate solution of aquatic weed biomass management in a sustainable approach.

Biohydrogen production by a novel strain Petroclostridium sp. X23 isolated from the production water of oil reservoirs

Biohydrogen production from H2-producing has been considered a promising alternative for the replacement of fossil fuels. However, the mechanisms that affect the H2-producing capacities of wild-type strains remain largely elusive. To explore the capabilities of biohydrogen production from microorganisms in the deep subsurface oil-reservoir environment, we isolated an H2-producing bacteria identified as the genus Petroclostridium from a long-term methanogenic crude oil-degrading enrichment culture of the production water from oil reservoir. The impact of various temperature, pH, and glucose concentrations on hydrogen production by Petroclostridium sp. X23 was investigated. Under the conditions of 37 °C, initial pH 8.0, and glucose concentration of 4 g/L, the strain X23 achieved its maximum hydrogen yield at 1.82 ± 0.12 mol H2/mol glucose. When compared to the previously reported strain SK-Y3, X23 exhibited enhanced H2-producing ability, with lesser energy biomass production. Comparative genomic analysis revealed that the genome of X23 contains more genes associated with hydrogen generation than SK-Y3. These findings proposed Petroclostridium sp. X23 is a potential candidate for dark fermentative hydrogen production under alkaline and mesophilic conditions. It also increased our knowledge of genotypes related to high H2-producing capability, which may provide instructions for future selection of high-yielding strains and reasonable metabolic engineering of wild-type H2-producing strains.

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.

Dark Fermentative Hydrogen Production from Spent Coffee Grounds Hydrolysate by Clostridium butyricum

Dark Fermentative Hydrogen Production from Spent Coffee Grounds Hydrolysate by Clostridium butyricum

Polydimethyldiallylammonium chloride inhibits dark fermentative hydrogen production from waste activated sludge

Polydimethyldiallylammonium chloride (PDDA) is an excellent flocculant for wastewater purification and sludge dewatering, but whether it poses a threat to hydrogen production from waste activated sludge is not known. In this study, the effect and underlying mechanism of PDDA on the dark fermentation of sludge was investigated. The results showed that PDDA reduced cumulative hydrogen production from 3.8±0.1 to 2.4±0.1 mL/g volatile suspended solids at 40 g/kg total suspended solids. PDDA impeded the dark fermentation process by inhibiting the activity of key enzymes, presenting a stronger inhibitory effect on the hydrogen production process than the hydrogen consumption process. Additionally, PDDA inhibited Firmicutes by enriching other microorganisms, thereby impeding hydrogen production via the acetate pathway. This study deepens the understanding of the potential effects of PDDA on sludge treatment and provides a theoretical basis for alleviating the negative effects of quaternary ammonium-based cationic flocculants.

Dark fermentative hydrogen production in packed-bed bioreactor using the Persian Gulf dead coral, ceramic saddle, and ceramic ball as support matrixes

Dark fermentative hydrogen production by Enterobacter aerogenes in a packed bed bioreactor using three immobilization supports was studied. The effects of glucose concentrations (20, 10, 5, 2.5, and 1.5 g/L) and support matrixes (Persian Gulf dead coral, ceramic saddle, and ceramic ball) on the hydrogen yield (HY) and hydrogen production rate (HPR) at low hydraulic retention time (HRT) were investigated. According to the achieved results, glucose concentration, porosity and shape support, and bed voidage affected hydrogen production. The HYs were increased with decreasing glucose concentration and at 2.5 g/L glucose concentration, the maximum HYs of 3.11, 2.86 and 2.79 mol hydrogen/mol glucose were obtained for dead coral, ceramic ball, and ceramic saddle, respectively. The results indicated that at the HRT of 0.6 h, the bioreactor packed with dead coral resulted in higher HY (3.11 mol H2/mol glucose) and HPR (173 mL/L.h) representing 8–11%, and 8–15%, enhancement compared to the bioreactor packed with ceramic supports. Besides, based on the acquired results, the HY and HPR of the ceramic ball were higher than the ceramic saddle.

Kinetic model derived from machine learning for accurate prediction of microalgal hydrogen production via conversion from low thermally pre-treated palm kernel expeller waste

The depletion of fossil fuel sources and increase in energy demands have increased the need for a sustainable alternative energy source. The ability to produce hydrogen from microalgae is generating a lot of attention in both academia and industry. Due to complex production procedures, the commercial production of microalgal biohydrogen is not yet practical. Developing the most optimum microalgal hydrogen production process is also very laborious and expensive as proven from the experimental measurement. Therefore, this research project intended to analyse the random time series dataset collected during microalgal hydrogen productions while using various low thermally pre-treated palm kernel expeller (PKE) waste via machine learning (ML) approach. The analysis of collected dataset allowed the derivation of an enhanced kinetic model based on the Gompertz model amidst the dark fermentative hydrogen production that integrated thermal pre-treatment duration as a function within the model. The optimum microalgal hydrogen production attained with the enhanced kinetic model was 387.1 mL/g microalgae after 6 days with 1 h thermally pre-treated PKE waste at 90 °C. The enhanced model also had better accuracy (R2 = 0.9556) and net energy ratio (NER) value (0.71) than previous studies. Finally, the NER could be further improved to 0.91 when the microalgal culture was reused, heralding the potential application of ML in optimizing the microalgal hydrogen production process.

Dark fermentative hydrogen production from cassava starch: A comprehensive evaluation of the effects of starch extrusion and enzymatic hydrolysis

Hydrolysis could be rate-limiting for the dark fermentative H2 production from cassava starch (CS) due to its semicrystalline nature. Therefore, the effects of CS extrusion and enzymatic hydrolysis on improving dark fermentation (DF) were studied. Preliminary DF experiments were conducted to investigate the effects of inoculum (sludge) heat-pretreatment, which increased H2 production from untreated-CS by over 154%. Likewise, 15 g/L starch was considered best for H2 production. Extruded-CS presented a maximum H2 yield of 1.58 mol H2/mol glucose with a 76% reduction in the fermentation adaptive phase and 43% increase in maximum H2 productivity compared to untreated-CS. Meanwhile, extruded-CS hydrolysate presented a maximum H2 yield of 1.8 mol H2/mol glucose with a 63% reduction in the fermentation adaptive phase and 18% increase in H2 productivity. A comprehensive assessment of the H2 yield, process time, production rate, and process simplification revealed extruded-CS as the most promising feedstock under the evaluated conditions.

Production of biohydrogen and green platform compound 2, 5-furandicarboxylic acid using rice straw hydrolysate

Lignocellulosic hydrolysate contains several inhibitors that hinder downstream biological processes. The immobilized strain Burkholderia sp. H-2 was used in this study to biodetoxify real rice straw hydrolysate and biotransform 5-hydroxymethylfurfural (5-HMF), an inhibitor, into 2, 5-furandicarboxylic acid (FDCA), a valuable platform compound. An electrodialysis with bipolar membranes (EDBM) system was applied to recover FDCA and perform secondary detoxification of the biodetoxified real rice straw hydrolysate. The rice straw hydrolysate after biodetoxification and EDBM treatment was used for anaerobic dark fermentative hydrogen production. The immobilized Burkholderia sp. H-2 could completely biotransform 1843 mg/L 5-HMF into 1532 mg/L FDCA within 24 h when the 2-fold diluted rice straw hydrolysate was used as the feedstock. The optimal EDBM system parameters to recover FDCA were a hydrolysate pH of 6, operated at a current density of 17.86 mA/cm2. The FDCA recovery efficiency was 44.6 %, when using the biodetoxified real rice straw hydrolysate as the EDBM feedstock. The rice straw hydrolysate after biodetoxification and EDBM treatment had better biohydrogen production performance than the undetoxified and biodetoxified rice straw hydrolysates.

Metal additives for boosting hydrogen production in anaerobic fermentation: Focus on the change of gene expression and cost analysis

Dark fermentation has emerged as a promising strategy for sustainable hydrogen production. However, the low hydrogen yield produced by anaerobic microbes continues to be a challenge. Although metal ion additives have been proposed to enhance hydrogen production, their entire impacts and underlying mechanisms are poorly understood. The present study aimed to examine the effects of Fe2+, Ni2+, Mg2+, and Mo6+ on dark fermentative hydrogen production, as well as their influences on microbial community analysis and gene expression in a batch system. The experiments were conducted under anaerobic conditions at 37 °C, utilizing 100 mM glucose as a substrate and an initial pH of 6.8. The results showed that Mo6+ at 0.015 mg-Mo6+/L significantly enhanced the hydrogen yield (2.21-fold increment) by upregulating nitrogenase gene expression (2.19-fold increment). Fe2+ at 50 mg-Fe2+/L was the second most effective metal ion, with a 1.81-fold increase in hydrogen yield and upregulation of ferredoxin (1.83-fold increment) and pyruvate formate-lyase gene (3.36-fold increment) expression. Mg2+ at 70 mg-Mg2+/L and Ni2+ at 25 mg-Ni2+/L also increased hydrogen yield by 1.51-fold and 1.31-fold, respectively, with upregulation of glyceraldehyde-3-phosphate dehydrogenase (1.02-fold and 1.81-fold increments, respectively) and ATP synthase (1.46-fold and 1.10-fold increments, respectively) expressions for both metal additives, pyruvate formate-lyase (2.77-fold increment) and pyruvate kinase (1.44-fold increment) gene expression for Mg2+. Regarding the mixed metal approach, it was observed that although certain combinations of mixed metals, such as Mg2++Mo6+, resulted in an enhancement of hydrogen yield by a factor of 1.37-fold, the increment was found to be lower than that of an individual metal. Therefore, the results of this study suggest that using a single metal would be more effective. Economic potential calculations further revealed that Mo6+ exhibited the highest economic feasibility, with the lowest expense of 2.02x10−5 $-metal/m3-H2, accounting for 0.002% of the average total production cost. This was followed by Mg2+, Fe2+, and Ni2+. Overall, our findings provide mechanistic insights into the use of metal ion additives for clean hydrogen production and offer a foundation for optimising their economic feasibility in large-scale hydrogen production.

Dark fermentative hydrogen production: Potential of food waste as future energy needs

Globally, food waste (FW) is found to be one of the major constituents creating several hurdles in waste management. On the other hand, the energy crisis is increasing and the limited fossil fuel resources available are not sufficient for energy needed for emerging population. In this context, biohydrogen production approach through valorization of FW is emerging as one of the sustainable and eco-friendly options. The present review explores FW sources, characteristics, and dark fermentative production of hydrogen along with its efficiency. FW are highly biodegradable and rich in carbohydrates which can be efficiently utilized by anaerobic bacteria. Based on the composition of FW, several pretreatment methods can be adapted to improve the bioavailability of the organics. By-products of dark fermentation are organic acids that can be integrated with several secondary bioprocesses. The versatility of secondary products is ranging from energy generation to biochemicals production. Integrated approaches facilitate in enhanced energy harvesting along with extended wastewater treatment. The review also discusses various parameters like pH, temperature, hydraulic retention time and nutrient supplementation to enhance the process efficiency of biohydrogen production. The application of solid-state fermentation (SSF) in dark fermentation improves the process efficiency. Dark fermentation as the key process for valorization and additional energy generating process can make FW the most suitable substrate for circular economy and waste based biorefinery.

Recent advances in process improvement of dark fermentative hydrogen production through metabolic engineering strategies

Biological means of hydrogen (H2) production has attracted tremendous research and development attention. Dark fermentation provides a possible way of producing H2 from a range of renewable energy sources, including wastewater. During fermentation, various metabolites are formed to create a complex metabolic flux network. Insufficient focus has been placed on the metabolic engineering that is intrinsic to fermentation. This current review summarizes the biochemical pathways occurring in the metabolic network of dark fermentation and how the key operational factors influence metabolism during dark fermentation. Recent developments and strategies for metabolic engineering that have been described to enhance H2 production are recommended. Finally, the economic analysis related to bio-H2 production and prospects is examined. It is envisaged that this study can give beneficial aspects in terms of fundamental knowledge, understanding, and the latest technology for scientists and research engineers in the field of bio-based H2 generation.

Supplementation of magnetic nanoparticles for enhancement of dark fermentative hydrogen production from pretreated garden wastes using Enterobacter aerogenes

The supplementation of magnetic nanoparticles (NPs) to the dark fermentative hydrogen production system mediated by Enterobacter aerogenes using pretreated garden wastes as feedstock was investigated to determine the enhancement of biohydrogen production efficiency. The optimized pretreatment conditions of 2% sulfuric acid with sonication (AS) and 2% Viscozyme L with sonication (ES) of garden wastes yielded a maximum of 33 and 37% total reducing sugar, respectively. In addition, the supplementation of 30 mg/L of magnetic NPs to dark fermentative system of AS and ES yielded 32 and 30% increased hydrogen production compared with the control without NPs, respectively. A maximum yield of 2.43 mol H2/mol sugar utilized was achieved in the optimized system supplemented with NPs. The reusability of the magnetic NPs offers an additional advantage showing more than 90% relative hydrogen production retained after four consecutive cycles. The proposed mechanisms suggest that the optimum level of magnetic NPs supplementation increases the electron transfer rate and modifies the E. aerogenes metabolic pathway, limiting ethanol production and increasing acetate proportion in the end metabolite analysis. Overall, the addition of magnetic NPs to the dark fermentative system improved the biological hydrogen production.

Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99µmol H2 g dry wt-1 and maximum hydrogenase activity of 179.39 ± 8.18µmol H2 g dry wt-1 min-1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Enhancing Dark Fermentative Hydrogen Production from Problematic Substrates via the Co-Fermentation Strategy

The aim of the present paper is the improvement of dark fermentative hydrogen production from problematic substrates. In detail, the study is aimed at (i) investigating the inhibiting effect of two problematic biomasses (i.e., of olive mill wastewater, containing recalcitrant/toxic compounds and cheese whey, lacking pH buffering capacity) on the dark fermentation process, (ii) as well as verifying the possibility to apply a co-fermentation strategy to enhance the process. To investigate the inhibiting effect of the substrates, two experimental sets were conducted using olive mill wastewater and cheese whey alone, under different food-to-microorganism ratios (i.e., 1, 2.5, and 5). Further experiments were conducted to verify the possibility of improving hydrogen production via the co-fermentation strategy. Such experiments included two tests conducted using different volumetric percentages of olive mill wastewater and cheese whey (90% olive mill wastewater + 10% cheese whey and 80% olive mill wastewater + 20% cheese whey). Results show that using olive mill wastewater alone, the inhibiting effect increased at a higher food-to-microorganism ratio. Moreover, because of the occurrence of a metabolic shift, hydrogen was not produced using 100% cheese whey. Interestingly, compared to the 100% olive mill wastewater condition, the use of 20% cheese whey allowed to double the hydrogen yield, reaching the high cumulative hydrogen production of 2.08 LL−1. Obtained results confirm that the two investigated substrates exert inhibiting effects on microorganisms. Nevertheless, co-fermentation is an effective strategy to improve the dark fermentation process of problematic biomass.

Pretreatment in Vortex Layer Apparatus Boosts Dark Fermentative Hydrogen Production from Cheese Whey

Dark fermentation (DF) is a promising process for mitigating environmental pollution and producing “green” H2. However, wider implementation and scaling of this technology is hampered by insufficient process efficiency. In this work, for the first time, the effect of innovative pretreatment of cheese whey (CW) in a vortex layer apparatus (VLA) on characteristics and DF of CW was studied. Pretreatment in VLA resulted in a heating of the CW, slight increase in pH, volatile fatty acids, iron, and reduction in fat, sugar, and chemical oxygen demand (COD). The biochemical hydrogen potential test and analysis of H2 production kinetics confirmed the significant potential of using VLA in enhancement of dark fermentative H2 production. The maximum potential H2 yield (202.4 mL H2/g COD or 3.4 mol H2/mol hexose) was obtained after pretreatment in VLA for 45 s and was 45.8% higher than the control. The maximum H2 production rate after 5 and 45 s of pretreatment was 256.5 and 237.2 mL H2/g COD/d, respectively, which is 8.06 and 7.46 times higher than in the control. The lag phase was more than halved as a function of the pretreatment time. The pretreatment time positively correlated with the total final concentration of Fe2+ and Fe3+ and negatively with the lag phase, indicating a positive effect of pretreatment in VLA on the start of H2 production.

New insights into mechanism of emerging pollutant polybrominated diphenyl ether inhibiting sludge dark fermentation

Polybrominated diphenyl ethers (PBDEs), derived from electronics, furniture, etc., are detected with high level in excess sludge (ES). In this work, the influence of PBDEs on ES dark fermentation (ESDF) hydrogen production and the related key mechanisms were explored. The result shows PBDEs exposure reduced hydrogen production, and hydrogen accumulation decreased from 17.6 mL/g in blank to 12.3 mL/g with 12.0 mg/Kg PBDEs. PBDEs induced the reactive oxygen species production, which directly led to cell inactivation and reduced hydrogen production. Furthermore, PBDEs decreased ES disintegration, hydrolysis, acidification and homoacetogenic processes and inhibited the activities of enzymes related to hydrogen production. PBDEs also affected the diversity and richness of microbial communities in dark fermentation systems, especially high doses of PBDEs reduced the relative abundance of microorganisms associated with hydrogen production. In conclusion, PBDEs reduce hydrogen generation from ES.

Thermophilic dark fermentation fast start-up of hydrogen production with substrate concentration regulation and moderate pretreatment inoculum

Accelerating start-up of thermophilic dark fermentation would improve hydrogen production efficiency was investigated. To find an efficient method for accelerating start-up and explore the effect of inoculum type for hydrogen production system, three thermophilic hydrogen production EGSB (expanded granular sludge bed) reactors equipped with flocculent or granular sludge using different startup modes were constructed in this study. Among three reactors under butyric acid type of fermentation, EGSB3 (startup mode: pretreated granular sludge as inoculum and regulated substrate concentration) was the most efficient one with shortest start-up time (13 days) and maximum hydrogen production (1.64 mol H2/mol glucose). EGSB1 (startup mode: pretreated flocculent sludge as inoculum and regulated substrate concentration) was started up in 27 days at 1.27 mol H2/mol glucose in maximum. While EGSB2 (startup mode: original granular sludge as inoculum and without regulating substrate concentration) took 46 days for start-up and the 1.6 mol H2/mol glucose hydrogen producing rate was obtained. After start-up, the dominant hydrogen production bacteria were Thermoanaerobacterium thermosaccharolyticum strain TG57 in three reactors. EGSB3 reactor owned the most proportion with 53.21 % than others. In all, mild pretreatment and regulating substrate concentration for granular sludge would be benefit for fast startup of thermophilic dark fermentation hydrogen production leading to shortest startup time, high hydrogen production efficiency and most functional microorganism.