Dark Fermentative Biohydrogen Production Research Articles

Recent advances in dark fermentative hydrogen production from vegetable waste: role of inoculum, consolidated bioprocessing, and machine learning.

Waste-centred-bioenergy generation have been garnering interest over the years due to environmental impact presented by fossil fuels. Waste generation is an unavoidable consequence of urbanization and population growth. Sustainable waste management techniques that are long term and environmentally benign are required to achieve sustainable development. Energy recovery from waste biomass via dark fermentative hydrogen production is a sustainable approach to waste management. Vegetable waste is generated in plenty over the food supply chain and being a rich source of carbon and other nutrients it has been studied for production of biohydrogen. This review aims to offer a comprehensive overview on the potential of vegetable waste as a feedstock for dark fermentative biohydrogen production. The hydrogen output from dark fermentative process is lower and additional strategies are required to improve the production. This review addresses the challenges generally encountered during dark fermentative hydrogen production using vegetable waste and the importance of methods such as bioaugmentation and application of extremophiles for process enhancement. The role of machine learning in the field of biohydrogen production is briefly discussed. The application of dark fermentative effluents for secondary valuable product generation and its contribution to the biohydrogen biorefinery is discussed as well.

Synergistic role of carbon quantum dots on biohydrogen production

Biohybrid system has their distinct ability to improve microbial fermentation. This study demonstrates the role of surface-doped carbon quantum dots (CQDs) on dark fermentative biohydrogen production using lactobacillus organic-hybrid biocatalyst. Herein, nitrogen-doped carbon quantum dots (N-CQDs) with < 5 nm (having a surface charge of +2.6 mV) and un-doped carbon quantum dots (CQDs) (having a surface charge of −4.5 mV) were synthesized via chemical assisted process. Subsequently, the biohybrid systems were constructed via augmentation of N-CQDs and CQDs with Lactobacillus delbreuckii and assessed for biohydrogen production. The results revealed that both the biohybrid systems (N-CQDs- Lactobacillus delbreuckii and CQDs- Lactobacillus delbreuckii) provided improved hydrogen production than that of the native bacterial strain. Interestingly, the obtained N-CQDs- Lactobacillus delbreuckii system provided the maximum hydrogen yield of 2.01 mol/mol hexose, followed by 1.86 mol/mol hexose from CQDs- Lactobacillus delbreuckii, which is about 33 % and 19 % higher than the bare bacterial strain. The electron transfer and metabolic alteration of microbes by carbon quantum dots were assessed using cyclic voltammetry (CV) and VFA production. It was concluded that the improved bio-hydrogen production from N-CQDs- Lactobacillus delbreuckii is attributed to enhanced electron transfer, which regulates the central metabolic pathway of acetate and butyrate synthesis with the least production of lactate and other reduced end product formation.

Efficient dark-fermentation biohydrogen production by Shigella flexneri SPD1 from biowaste-derived sugars through green solvents approach

This study evaluated the dark-fermentative hydrogen (H2) production potential of isolated and identified Shigella flexneri SPD1 from various pure (glucose, fructose, sucrose, lactose, and galactose) and biowastes (coconut coir, cotton fiber, groundnut shells, rice-, and wheat-straws)-derived sugars. Among sugars, S. flexneri SPD1 exhibited high H2 production of up to 3.20 mol/mole of hexose using glucose (5.0 g/L). The pre-treatment of various biowastes using green solvents (choline chloride and lactic acid mixture) and enzymatic hydrolysis resulted in the generation of up to 36.0 g/L of sugars. The maximum H2 production is achieved up to 2.92 mol/mol of hexose using cotton-hydrolysate. Further, the upscaling of bioprocess up to 5 L of capacity resulted in a maximum yield of up to 3.06 mol/mol of hexose. These findings suggested that S. flexneri SPD1, a novel H2-producer, can be employed to develop a circular economy-based approach to produce clean energy.

Rederiving kinetics to model biohydrogen production from immobilized microalgae alginate beads at various polymerization degrees of alginate under dark fermentative environment

The performance of immobilized microalgae-alginate beads on biohydrogen production and its stability across several dark fermentation cycles is influenced by the sodium alginate concentrations. Thus, it is vital to determine the optimal condition for immobilization to achieve maximum encapsulation efficiency, stability and biohydrogen production. In this work, different sodium alginate concentrations (2 to 8 w/v%) were used to immobilize microalgae in influencing the dark fermentative biohydrogen productions from municipal wastewater, and its reusability was also investigated. The immobilized microalgae-alginate beads with sodium alginate concentration of 2 % showed the lowest stability and biohydrogen production in all cycles. The highest biohydrogen production was achieved by immobilized microalgal-alginate beads prepared from 8 % sodium alginate, followed by 6 % and 4 % in the first and second cycles. However, 8 % of immobilized microalgae-alginate beads generated the lowest biohydrogen volume during the third cycle. Besides, a model was rederived from the modified Gompertz model and Fick's law of diffusion equation to describe the relationship between polymeric viscosity and biohydrogen production from immobilized microalgae-alginate beads with different sodium alginate concentrations. As the sodium alginate concentrations increased, the viscosity also increased which significantly affected the properties of immobilization matrix formed such as encapsulation efficiency, growth of microalgae, diffusion of substrate and biohydrogen yield. The rederived model managed to fit the experimental data with a coefficient of determination values of >0.95 for all the polymerization degrees of alginate. The kinetic parameters, namely, yield of biohydrogen and specific microalgae growth rate of sodium alginate concentration of 6 % were 129.80 L kg−1 and 4.69 h−1, respectively, which were considered as maximum results as compared with other sodium alginate concentrations. Besides, the difference in biohydrogen productions for all cycles and kinetic data of biohydrogen yields obtained from the rederived model between sodium alginate concentration of 6 % and 8 % only exhibited a slight variance (<3 %). Thus, sodium alginate concentration of 6 % was considered to be an optimal for immobilizing microalgae in performing the dark fermentation. Overall, the results revealed the significance of suitable sodium alginate concentration in maximizing the immobilization of microalgae for performing the dark fermentative biohydrogen production.

Metabolic and process engineering of Clostridium tyrobutyricum for efficient hydrogen production from sugarcane molasses

Clostridia is the dominant microorganism in dark-fermentative biohydrogen production, but there have been relatively few studies on hydrogen production by Clostridium tyrobutyricum using cheap biomass. In this study, the important process parameters for hydrogen fermentation, such as substrate concentration, pH and temperature, were optimized systematically. Then, the effect of endogenous hydrogenases overexpression on hydrogen production was studied. To achieve economical hydrogen production from cheap substrate sugarcane molasses, the sucrose metabolic pathway was further introduced into C. tyrobutyricum. As a result, the engineered C. tyrobutyricum produced 1098.4 mL hydrogen with a yield of 248.2 mL/g sugar and productivity of 199.6 mL/L/h under the optimum fermentation conditions, which is the highest hydrogen production (910.37 mmol/L) reported so far using molasses. This study demonstrates that C. tyrobutyricum is a promising cell factory for hydrogen production from cheap and renewable biomass resources.

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.

A bibliometric analysis of the role of nanotechnology in dark fermentative biohydrogen production.

Recently, nanoparticles have drawn a lot of interest as catalysts to enhance the effectiveness and output of biohydrogen generation processes. This review article provides a comprehensive bibliometric analysis of the significance of nanotechnology in dark fermentative biohydrogen production. The study examines the scientific literature from the database of The Web of Science© while the bibliometric investigation utilized VOSviewer© and Bibliometrix software tools to conduct the analysis. The findings revealed that a total of 232 articles focused on studying dark fermentation for hydrogen production throughout the entire duration. The extracted data was used to analyze publication trends, authorship patterns, and geographic distribution along with types and effects of nanoparticles on the microbial community responsible for dark fermentative biohydrogen production. The findings of this bibliometric analysis provide valuable insights into the advancements and achievements in the utilization of nanoparticles in the dark fermentation process used to produce biohydrogen.

Unravelling the kinetics, isotherms, thermodynamics, and mass transfer behaviours of Zeolite Socony Mobil - 5 in removing hydrogen sulphide resulting from a dark fermentative biohydrogen production process.

Research into the speciation of sulfur and hydrogen molecules produced through the complex process of thermophilic dark fermentation has been conducted. Detailed surface studies of solid-gas systems using real biogas (biohydrogen) streams have unveiled the mechanisms and specific interactions between these gases and the physicochemical properties of a zeolite as an adsorbent. These findings highlight the potential of zeolites to effectively capture and interact with these molecules. In this study, the hydrogen sulphide removal analysis was conducted using 0.8 g of the adsorbent and at various reaction temperatures (25-125 °C), a flow rate of 100 mL min-1, and an initial concentration of approximately 5000 ppm hydrogen sulphide. The reaction temperature has been observed to be an essential parameter of Zeolite Socony Mobil - 5 adsorption capacity. The optimum adsorption capacity attains a maximum value of 0.00890 mg g-1 at an optimal temperature of 25 °C. The formation of sulphur species resulting from the hydrogen sulphide adsorption on the zeolite determines the kinetics, thermodynamics, and mass transfer behaviours of Zeolite Socony Mobil - 5 in hydrogen sulphide removal and Zeolite Socony Mobil - 5 is found to improve the quality of biohydrogen produced in thermophilic environments. Biohydrogen (raw gas) yield was enhanced from 2.48 mol H2 mol-1 hexose consumed before adsorption to 2.59 mol H2 mol-1 hexose consumed after adsorption at a temperature of 25 °C. The Avrami kinetic model was fitted for hydrogen sulphide removal on Zeolite Socony Mobil - 5. The process is explained well and fitted using the Temkin isotherm model and the investigation into thermodynamics reveals that the adsorption behaviour is exothermic and non-spontaneous. Furthermore, the gas molecule's freedom of movement becomes random. The adsorption phase is restricted by intra-particle diffusion followed by film diffusion during the transfer of hydrogen sulphide into the pores of Zeolite Socony Mobil - 5 prior to adsorption on its active sites. The utilisation of Zeolite Socony Mobil - 5 for hydrogen sulphide removal offers the benefit of reducing environmental contamination and exhibiting significant applications in industrial operations.

Comparative effect of acid and heat inoculum pretreatment on dark fermentative biohydrogen production

This study delves into the impact of various pretreatment methods on the inoculum in dark fermentation trials, specifically exploring thermal shock at different temperatures (60, 80, and 100 °C) and durations (15, 30, and 60 min), as well as acid shock at pH 5.5. Initial acidification of the substrate/inoculum mixture facilitates H2 generation, making acid shock an effective pretreatment option. However, it is also observed that combining thermal and acid pretreatments boosts H2 production synergistically. The synergy between thermal and acid pretreatments results in a significant improvement, increasing the overall hydrogen production efficiency by more than 9% compared to assays involving acidification alone. This highlights the considerable potential for optimizing pretreatment strategies. Furthermore, the study sheds light on the critical role of inoculum characteristics in the process, with diverse hydrogen-generating bacteria significantly influencing outcomes. The established equivalent performance of HCl and H2SO4 in inoculum pretreatment demonstrates the versatility of these acids in shaping the microbial community and influencing hydrogen production. The analysis of glucose conversion data highlights a prevalence of butyric acid in all trials, irrespective of the pretreatment method, emphasizing the dominance of the butyrate pathway in hydrogen generation. Additionally, an examination of the microbial community offers valuable insights into the intricate relationships between temperature, pH, and microbial diversity. Bacteroidota established its dominance among the bacterial populations, with a relative abundance exceeding 20–25% in the raw inoculum, and this dominance further increased following the treatment. Thermal and acid pretreatments result in significant shifts in dominant microbial communities, with some non-dominant phyla like Cloacimonadota and Spirochaetota becoming more prominent. These shifts in microbial diversity underscore the sensitivity of microbial communities to environmental conditions and pretreatment methods, further highlighting the importance of understanding their dynamics in dark fermentation processes.

Screening of microalgae strains for efficient biotransformation of small molecular organic acids from dark fermentation biohydrogen production wastewater

Dark fermentation biohydrogen production is a rapidly advancing and well-established field. However, the accumulation of volatile organic acid (VFAs) byproducts hinder its practical applications. Microalgae have demonstrated the ability to efficiently utilize VFAs while also treating waste gases and other nutrient elements. Integrating microalgae cultivation with dark fermentation is a promising approach. However, low VFAs tolerance and slow VFAs consumption restrict their application. To find suitable wastewater treatment microalgae, this work screened eight microalgae strains from five family. The results demonstrated that Chlamydomonas reinhardtii exhibited significant advantages in VFAs utilization, achieving a maximum removal of 100% for acetate and 52.5% for butyrate. Among the tested microalgae strains, CW15 outperformed in terms of photobioreactor adaptability, VFAs utilization, biomass productivity, and nutrient removal, making it the most promising microalgae for practical applications. This research demonstrates the feasibility of integrating microalgae cultivation with dark fermentation and providing a viable technical solution for integrated-biorefining.

Comparison of biorefinery characteristics: Photo-fermentation biohydrogen, dark fermentation biohydrogen, biomethane, and bioethanol production

Biorefinery technology promotes the realization of the Paris Agreement, China’s achievement of its carbon peak, and carbon neutralization. Photo-fermentation biohydrogen production, dark fermentation biohydrogen production, biomethane production, and bioethanol production are several promising biorefinery methods. In this study, corn stover was used as the raw material, and the gas, liquid, and kinetic characteristics of four biorefinery methods were investigated. The production yield, gross thermal energy, economy, and greenhouse gas emissions of the target product per gram of corn stover were studied. The maximum yields of photo-fermentation biohydrogen production, dark fermentation biohydrogen production, biomethane production, and bioethanol production were 68.4 mL/g dry matter (DM), 47.4 mL/g DM, 196.0 mL/g DM, and 2.7 g/L, respectively. The maximum gross thermal energy obtained from biomethane production was 7017.3 J/g DM. The maximum economy of 1.4 RMB/g DM was observed in photo-fermentation biohydrogen production. The highest greenhouse gas emission of the target product from biomethane production was 196.0 mL/g DM. The results provide a reference for the efficient utilization of biomass resources and a comprehensive evaluation of biorefinery technology.

Effect of nanoparticles synthesized from green extracts on dark fermentative biohydrogen production

Research on the production of nanoparticles with sustainable methods using different leaf extracts as a reducing agent has gained momentum. In this study, green synthesis of two types of iron oxide nanoparticles were produced to investigate the impact of nanoparticles on bio-hydrogen yield. Olive leaf extract and microalgae extract were used as reducing agents for production. The synthesized nanoparticles were characterized via SEM, EDX, XRD and UV–Visible spectrum analyses. The nanoparticles have a stable appearance and mean particle sizes of 90.26 and 124.68 nm. Iron oxide nanoparticles produced from olive leaf extract were more effective in dark fermentation. Dark fermentation yields by Clostridium sp. were assessed utilizing various proportions of generated two types of nanoparticles (50–400 mg/L). The addition of 200 mg/L olive leaf extract-based iron oxide nanoparticles and 300 mg/L microalgae extract-based iron oxide nanoparticles enhanced the hydrogen production capacity by 41% and 28%, respectively.

Optimization of dark fermentative biohydrogen production from rice starch by Enterobacter aerogenes MTCC 2822 and Clostridium acetobutylicum MTCC 11274.

Dark fermentative biohydrogen production (DFBHP) has potential for utilization of rice starch wastewater (RSWW) as substrate. The hydrogen production of Enterobacter aerogenes MTCC 2822 and Clostridium acetobutylicum MTCC 11274, in pure culture and co-culture modes, was evaluated. The experiments were performed in a 2L bioreactor, for a batch time of 120h. The co-culture system resulted in highest cumulative hydrogen (1.13L H2/L media) and highest yield (1.67mol H2/mol glucose). Two parameters were optimized through response surface methodology (RSM)-substrate concentration (3.0-5.0g/L) and initial pH (5.5-7.5), in a three-level factorial design. A total of 11 runs were performed in duplicate, which revealed that 4.0g/L substrate concentration and 6.5 initial pH were optimal in producing hydrogen. The metabolites produced were acetic, butyric, propionic, lactic and isobutyric acids. The volumetric H2 productions, without and with pH adjustments, were 1.24L H2/L media and 1.45L H2/L media, respectively.

Effect of bioaugmentation using Clostridium butyricum on the start-up and the performance of continuous biohydrogen production

This study aimed to mitigate the instability in the start-up and continuous performance of dark fermentative biohydrogen production using heat-treated sludge by the addition of an exogenous H2-producing strain. Continuous fermentation augmented with Clostridium butyricum showed the highest average biohydrogen production rate (HPR) as 50.35 ± 2.56 and 58.57 ± 5.03 L/L-d with H2-producing butyric and acetic acid pathways, whereas the fermenters without bioaugmentation showed the termination of biohydrogen production in 3 days of continuous operation with non H2-producing lactic acid pathway and H2-consuming propionic acid pathway. The bioaugmentation blocked the growth of the competitors for hexose such as Streptococcus, Lactobacillus and Megasphaera, and provided H2-producer dominated microbiome with not only Clostridium butyricum, but also Clostridium puniceum and Clostridium neuense originated from heat-treated sludge. Bioaugmentation of a H2-producing strain would be a reliable dissemination strategy for dark fermentative biohydrogen production by minimizing the influence of seed sludge population.

Bio-Hydrogen Production in Packed Bed Continuous Plug Flow Reactor—CFD-Multiphase Modelling

This research study investigates the modelling and simulation of biomass anaerobic dark fermentation in bio-hydrogen production in a continuous plug flow reactor. A CFD multiphase full transient model in long-term horizons was adopted to model dark fermentation biohydrogen production in continuous mode. Both the continuous discharge of biomass, which prevents the accumulation of solid parts, and the recirculation of the liquid phase ensure constant access to the nutrient solution. The effect of the hydraulic retention time (HRT), pH and the feed rate on the bio-hydrogen yield and production rates were examined in the simulation stage. Metabolite proportions (VFA: acetic, propionic, butyric) constitute important parameters influencing the bio-hydrogen production efficiency. The model of substrate inhibition on bio-hydrogen production from glucose by attached cells of the microorganism T. neapolitana applied to the modelling of the kinetics of bio-hydrogen production was used. The modelling and simulation of a continuous plug flow (bio)reactor in biohydrogen production is an important part of the process design, modelling and optimization of the biological H2 production pathway.

Novel strategies towards efficient molecular biohydrogen production by dark fermentative mechanism: present progress and future perspective.

In the scenario of alarming increase in greenhouse and toxic gas emissions from the burning of conventional fuels, it is high time that the population drifts towards alternative fuel usage to obviate pollution. Hydrogen is an environment-friendly biofuel with high energy content. Several production methods exist to produce hydrogen, but the least energy intensive processes are the fermentative biohydrogen techniques. Dark fermentative biohydrogen production (DFBHP) is a value-added, less energy-consuming process to generate biohydrogen. In this process, biohydrogen can be produced from sugars as well as complex substrates that are generally considered as organic waste. Yet, the process is constrained by many factors such as low hydrogen yield, incomplete conversion of substrates, accumulation of volatile fatty acids which lead to the drop of the system pH resulting in hindered growth and hydrogen production by the bacteria. To circumvent these drawbacks, researchers have come up with several strategies that improve the yield of DFBHP process. These strategies can be classified as preliminary methodologies concerned with the process optimization and the latter that deals with pretreatment of substrate and seed sludge, bioaugmentation, co-culture of bacteria, supplementation of additives, bioreactor design considerations, metabolic engineering, nanotechnology, immobilization of bacteria, etc. This review sums up some of the improvement techniques that profoundly enhance the biohydrogen productivity in a DFBHP process.

Dark fermentative biohydrogen production from confectionery wastewater in continuous-flow reactors

The production of biohydrogen from industrial wastewater through the dark fermentation (DF) process has attracted increased interest in recent years. To implement a DF process on a large scale, a thorough knowledge of laboratory scale process control is required. The operating parameters and design features of the reactors have a great influence on the efficiency of the process. In this work, the possibility of continuous production of biohydrogen from confectionery wastewater was evaluated. The DF process was carried out at 37 ± 1 °C in two different reactors: an upflow anaerobic filter (AF) and a fluidized bed reactor (AFB). Polyurethane foam (PU) was used to immobilize the biomass. The DF process was studied at four hydraulic retention times (HRT) (1.5, 2.5, 7.5 and 15 days) and the corresponding organic loading rates (OLR) (9.21, 6.12, 2.04 and 1.02 g CODinit/(L day)). The highest hydrogen yield (HY) (44.73 ml/g CODinit) and hydrogen production rate (HPR) (92.5 ml/(L day)) was observed in AFB at HRT of 7.5 days and 2.5 days, respectively. The highest concentration of hydrogen in biogas was 34% in AF and 36% in AFB at HRT of 7.5 days. In contrast to AF, the COD removal efficiency in AFB increased with increasing HRT. The pH of the effluent was low (3.95–4.38). However, due to the use of PU for biomass immobilization, it is possible that there were local zones in the reactor that were optimal for the functioning of not only acidogens, but also methanogens. This was evidenced by a rather high content of methane in biogas (2.5% in AF and 9.6% in AFB at HRT of 15 days). These results provide valuable data for optimizing the continuous DF of wastewater from confectionery and other food industries to produce biohydrogen or biohythane.

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H 2 /g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production. • Cladophora sp. macroalgae was used as feedstock for biohydrogen production. • H 2 production performance of various I/S ratios was investigated. • The maximum hydrogen yield of 54.7 ml H 2 /g VS was achieved. • Mathematical models were used to determine kinetic parameters of H 2 production.

Scale-Up of Dark Fermentative Biohydrogen Production by Artificial Microbial Co-Cultures

As a renewable energy carrier, dark fermentative biohydrogen (H2) represents a promising future alternative to fossil fuels. Recently, the limited H2 yield of 4 moles of H2 per mole glucose, the so-called “Thauer limit”, was surpassed by a defined artificial consortium. In this article, we demonstrate the upscaling of this drawing board design, from serum bottles to laboratory scale bioreactors. Our results illustrate that this designed microbial co-culture can be successfully implemented in batch mode, with maximum H2 yields of 6.18 and 4.45 mol mol−1 substrate. Furthermore, we report volumetric H2 productivities of 105.6 and 80.8 mmol H2 L−1 h−1. These rates are higher than for any other dark fermentative H2 production system using a synthetic microbial co-culture applied in batch mode on a defined medium. Our study is an important step forward for the application of artificial microbial consortia in future biotechnology and energy production systems.

Dark fermentative biohydrogen production from vinicultural biomass without exogenous inoculum in a semi-batch reactor: A kinetic study

This work employed a unique kind of vinicultural biomass (grape residues) to generate fermentative hydrogen. This form of biomass serves two purposes (contains substrate and inoculum). Four mathematical model methods were established; these models were used to represent the fluctuation of hydrogen generation and other fermentation products (organic acids, alcohols), the consumption of substrates included in biomass, and bacterial growth. One of these models was verified using experimental data and used to represent all of the metabolic pathways of bacteria contained in the medium and the interaction between products and substrates.The optimal biomass load, 60 g COD (Chemical Oxygen Demand)/L with a concentration of 0.22 mol of hexose and 0.0444 mol of tartrate offers the best hydrogen yield.