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 H₂/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.     

Feasible pretreatment of textile wastewater for dark fermentative hydrogen production

In this study, the yield of hydrogen production was investigated under different feedstock pretreatment conditions. The feedstock for dark fermentative hydrogen production was textile wastewater which was obtained from the de-sizing process in a textile factory, located in northern Taiwan. The wastewater was pretreated with activated carbon, cation exchange resin or was not pretreated before being fed into the batch bottles. Biohydrogen production was carried out in a batch reactor with the sludge of mixed-culture using the feedstock from the pretreated wastewater. The sludge was obtained from the Taichung municipal wastewater treatment plant. The yield of hydrogen production using the two pretreatment methods or non – treatment were compared.     

An efficient dark fermentative hydrogen production by GMV control of pH

This study proposes that the on-line pH control via a model-based adaptive controller markedly improves the dark fermentative hydrogen production. According to the dynamic behavior of the dark fermentation process, pH, which rapidly declines with the beginning of the biogas production, should be precisely controlled around its optimal value in a narrow range. The success of on-line pH control was guaranteed by performing the preliminary simulation studies by experimental data obtained from dynamic analysis to determine ARMAX model order with Recursive Least Squares parameter estimation method and then to control the pH with Generalized Minimum Variance (GMV) controller. On-line control of pH at the optimal value of 6.0 during the 25 h dark fermentation process resulted in 5.4 times higher biogas production, 6.2 times higher biogas production potential, nearly doubled the duration of fermentation, and 18.4% biogas production rate increment in comparison with the uncontrolled pH case.     

Changes in microbial community structure during dark fermentative hydrogen production

Microbial community structure plays a significant role in the efficiency of dark fermentative hydrogen production using mixed culture. However, the detailed evolutions in microbial community structure during dark fermentation process are still unclear. This study investigated the detailed evolution patterns of microbial community structure during dark fermentation process by high-throughput pyrosequencing. Results showed that microbial community structure changed significantly over time in dark fermentation. Microbial diversity showed a constant decreasing trend during the fermentation process. The analysis of microbial community composition showed that Clostridium sensu stricto 1, Paraclostridium, Romboutsia and Paeniclostridium, which were all rarely existed in the inoculum, dramatically became dominant genera in the system after 6 h fermentation, with total relative abundance of more than 99%. This interesting result revealed that how quickly hydrogen-producing genera overwhelmed the microbial community in dark fermentation. Spearman correlation analysis showed that Clostridium sensu stricto 1 contributed the most to hydrogen fermentation performances.     

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

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

Effect of enzyme addition on fermentative hydrogen production from wheat straw

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H₂), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H₂ usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H₂ production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H₂ production from untreated wheat straw ranged from 5.18 to 10.52 mL-H₂ g-VS⁻¹. Whatever the experimental enzyme addition procedure, a two-fold increase in H₂ production yields ranging from 11.06 to 19.63 mL-H₂ g-VS⁻¹ was observed after enzymatic treatment of the wheat straw. The high variability in H₂ yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H₂ production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw⁻¹.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.     

Optimization and modelling of dark fermentative hydrogen production from cheese whey by Enterobacter aerogenes 2822

In India, annually about 3.3–5 million tons of cheese whey is produced which may causes serious problems for the environment if left untreated. In this study, pretreated cheese whey was utilized to produce hydrogen via dark fermentation by Enterobacter aerogenes 2822 cells in 2 L double walled cylindrical bioreactor having working volume of 1.5 L. Effect of change in total carbohydrate concentration in cheese whey (CW_TC, 20–45 g L^?1), temperature (T, 25–37 °C) and pH (5.5–7.5) was investigated on volumetric hydrogen production rate (VHPR) using Box Behnken design (BBD). Experimental VHPR of 24.7 mL L^?1 h^?1 was attained at an optimum concentration of 32.5 g L^?1 CW_TC, 31 °C T and 6.5 pH, which was in good correlation with predicted rate of 23.2 mL L^?1 h^?1. Mathematical models based on Monod and logistic equations were developed to describe the kinetics of substrate consumption and growth profile of E. aerogenes 2822 under optimum conditions. While for the modelling of fermentative hydrogen production in batch mode, Modified Gompertz equation and Leudeking-Piret models were used which gave proper simulated fitting. These results will add significant values to cheese whey by converting it into a clean form of bioenergy.     

Comparison of fermentative hydrogen production from glycerol using immobilized and suspended mixed cultures

This study explored the fermentative hydrogen production by immobilized microorganisms from glycerol, which is the byproduct of biodiesel production, and compared it with suspended fermentation. The effect of immobilization on hydrogen production process was examined. Results showed that both cumulative hydrogen production (CHP) and hydrogen yield (HY) were enhanced by microbial immobilization. The highest CHP and HY of 64 mL/100 mL and 0.52 mol H₂/mol glycerol were obtained by immobilized microorganisms, compared to 9 mL/100 mL and 0.29 mol H₂/mol glycerol in suspended microorganisms. Immobilization enhanced CHP and HY by 611.1% and 79.3%. In addition, immobilized microorganisms showed stronger tolerance to high substrate concentration and higher capability in glycerol utilization, which is of great significance for hydrogen production from glycerol. The enhanced hydrogen production may be due to the favorable micro-environment for different microorganisms in immobilized beads.     

Auto-selection of microorganisms of sewage sludge used as an inoculum for fermentative hydrogen production from different substrates

When mixed organic waste is used for hydrogen production by dark fermentation, the microbial community which is most adapted to the actual biopolymer composition of the substrate is auto-selected. In this research, six substrates simulating different biopolymers (proteins, fats, carbohydrates) and their mixtures were used to enrich hydrogen-producing bacteria adapted to these substrates from non-pretreated sewage sludge. Phylum Firmicutes dominated in the microbial community (67–100%) regardless of the substrate used, as was shown by high-throughput sequencing. Microbial diversity was low when using carbohydrate-rich substrates and the microbial community was mainly represented by Ruminococcus (26–90%) and Thermoanaerobacterium (6–67%). Dark fermentation of fats and proteins was characterized by higher microbial diversity. Thermoanaerobacterium (21%), Thermobrachium (19%), Tepidiphilus (16%) and Acetomicrobium (14%) dominated when using fats, while Thermobrachium (34%), Acetomicrobium (16%) and Clostridium sensu stricto 7 (12%) dominated when using proteins, as substrate. Different microbial communities and substrates resulted in diverse process performance and metabolic pathways. Dark fermentation of starch achieved the maximum hydrogen yield of 138 mL/g volatile solids with 60.4% hydrogen content in biogas. The dominance of genus Ruminococcus was thought to be responsible for the highest hydrogen production. Minor quantities of methane from proteins and fats were produced by Methanothermobacter and Methanosarcina. Based upon the stable ¹³C isotope analysis, the hydrogenotrophic pathway was a slightly more predominant methane formation route than the others considered.     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.     

Dark fermentative hydrogen production from food waste: Effect of biomass ash supplementation

This work aimed to investigate the effects of supplementing two distinct types of ash, namely fly ash (FA) and bottom ash (BA) on the dark fermentation (DF) process of food waste (FW) for H₂ production. Both types of biomass combustion ash (BCA) were collected in an industrial bubbling fluidized bed combustor, using residual forest biomass as fuel. Results indicated that adding BCA at different doses of 1, 2 and 4 g/L could effectively enhance H₂ generation when compared to the control test without BCA addition. This stimulatory effect was attributed to the crucial role of metal elements released from BCA such as sodium, potassium, calcium, magnesium, and iron in the provision of buffering capacity and inorganic nutrients for the functioning of hydrogen-forming bacteria. The highest H₂ yield of 169 mL per g of volatile solids (VS) were obtained by adding only a small amount of BA (1 g/L) to the reactive system, representing a significant increment of 1070% compared to the control reactor. Furthermore, a significant decrease in the bacterial lag phase time from 26 h to 2.7 h, as well as about a 12-fold increase in the energy recovery as H₂ gas was observed at BA dosage of 1 g/L in comparison with the control reactor. Overall, this study suggested that a proper addition of BCA could promote the DF process of FW and enhance biohydrogen production.     

The CRISPR technology: A promising strategy for improving dark fermentative biohydrogen production using Clostridium spp.

Generating hydrogen gas (H₂) using the dark fermentation method has attracted much attention due to its lower energy requirement and environmental friendliness. However, producing a high yield of bio-H₂ is as challenging as ever due to low energy conversion by microorganisms. In this respect, the advancement of genome editing tools including the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology could overcome the established maximum ceiling of product yield. To date, CRISPR-Cas systems, particularly those based on Type II CRISPR-Cas9 and Type V CRISPR-Cas12, are widely used in manipulating novel bacteria to improve the yield of specific biofuel. However, studies using the CRISPR-Cas technology for improving bio-H₂ production remain scarce. Understanding the metabolic pathways of Clostridium spp. is essential for using the CRISPR-Cas technology Thus, this review highlighted the state-of-the-art in CRISPR-Cas systems for bacterial genome editing while paying attention to bioprocess optimization strategies for modulating the biohydrogen production.     

Biohydrogen yield efficiency and the benefits of dark,photo and dark-photo fermentative production technology in circular Asian economies

Twenty-six new data envelopment analysis (DEA) models with 55 biohydrogen production experiments categorized into three groups including dark fermentation (DF), photo fermentation (PF), and dark-photo sequential fermentation (DF-PF) technologies, are used to evaluate their biohydrogen yield efficiency. The results reveal the average yield efficiencies of DF, PF and DF-PF are 0.2844, 0.3460 and 0.7040, respectively. The most efficient overall combination of biohydrogen inputs is PhBR1/Rhodobacter capsulatus B10/Rhodobacter capsulatus in DF-PF. Statistical tests demonstrate DF-PF has statistically double the efficiency of PF and DF, and the efficiency of PF significantly exceeds that of DF, supporting some of the literature findings. A flexible DEA model must be carefully chosen when evaluating biohydrogen production. All inputs and outputs of biohydrogen statistically influenced yield efficiency to a significant level. India and Japan are the top two economies benefitting from improved biohydrogen yield efficiency. Improving biohydrogen yield efficiency can improve macroeconomic growth and develop the renewable hydrogen and biohydrogen industry.     

Impact of the microbial inoculum source on pre-treatment efficiency for fermentative H2 production from glycerol

Hydrogen (H₂) production by dark fermentation can be performed from a wide variety of microbial inoculum sources, which are generally pre-treated to eliminate the activity of H₂-consuming species and/or enrich the microbial community with H₂-producing bacteria. This paper aims to study the impact of the microbial inoculum source on pre-treatment behavior, with a special focus on microbial community changes. Two inocula (aerobic and anaerobic sludge) and two pre-treatments (aeration and heat shock) were investigated using glycerol as substrate during a continuous operation. Our results show that the inoculum source significantly affected the pre-treatment efficiency. In aerobic sludge no pre-treatment is necessary, while in anaerobic sludge the heat pre-treatment increased H₂ production but aeration caused unstable H₂ production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and unstable H₂ production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H₂ production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.     

Biohydrogen production from acid hydrolyzed wastewater treatment sludge by dark fermentation

Waste generation, waste management, sustainable energy production, and global warming are interrelated environmental issues to be considered together. Wastewater treatment sludge is an organic substance rich waste which causes significant environmental problems. However, these wastes can be used as raw material in biofuel generation. This study was designed to investigate the possible utilization of waste sludge in biohydrogen production by taking these facts into consideration. For this purpose, the sludge was first pre-treated with acid and then, the solid (sludge) and liquid (filtrate) phases of acid pre-treated sludge were used as the substrates for biohydrogen generation dark fermentation. Two-factor factorial experimental design method was used in acid hydrolysis of sludge to determine the effect of pH (pH = 2–6) and reaction period (time, min) elution of chemical oxygen demand (COD), total organic carbon (TOC) and total sugar (TS), NH₄N and PO₄P. Statistical evaluation of the results indicated that pH significantly affects the elution of organic carbon and nutrient content of sludge while the reaction time is significant for only organic carbon content. The optimum pretreatment conditions for maximum organic and nutrient elution were determined as pH = 2 and t = 1440 min. The pretreated products, named as filtrate sludge and sludge, conducted to dark fermentation under mesophilic conditions for biohydrogen generation showed that pretreatment of waste sludge at pH = 6 is the best condition giving the maximum yields (Y_H2) as Y_H2 = 24 mmol g⁻¹ Total Sugar _consumed and Y_H2 = 41 mmol g⁻¹ Total sugar consumed, for filtrate and sludge, respectively.     

Enhanced dark fermentative hydrogen production under the effect of zero-valent iron shavings

Dark fermentative hydrogen production under the effect of zero-valent metal shavings (iron, aluminum and copper) was studied by using a sucrose medium and a mixed bacterial consortium. The iron shavings were found to be unique to promote the hydrogen production, the hydrogen yield obtained from an optimal dose of 8–16 g/L reached 4.2 mol/mol hexose, doubled compared with that obtained from the control without addition of the iron shavings. The effect was more obvious in low pH buffered medium than in higher buffered medium. The aluminum and copper shavings were either inert or toxic to the cultivation. It is evident that the addition of the zero-valent iron helped maintaining the pH to an optimal range for hydrogen production and drove more reducing equivalents to the production of hydrogen. A microbial corrosion system mediated by the hydrogen producing bacteria was proposed to be responsible for the improvement of hydrogen production.