Enhancement of fermentative biohydrogen production from textile desizing wastewater via coagulation-pretreatment

A real textile desizing wastewater (TDW) was coagulation-pretreated to enhance its potential of biohydrogen production. Batch fermentation showed that the hydrogen production was efficiently enhanced (550 and 120% increments for hydrogen production rate and hydrogen yield, respectively) and the production performance was substrate-concentration dependent. A peak hydrogen production rate of 3.9 L/L-d and hydrogen yield of 1.52 mol/mol hexose were obtained while using coagulant GGEFloc-653 at a dosage of 1 g/L to pretreat TDW with the concentration of 15 g total sugar/L. The coagulation-pretreatment could have butyrate-type fermentation with high biohydrogen production and the removed some toxic materials that might drive the metabolic pathways to those not favoring biohydrogen production. Based on the data obtained, strategies to operate the coagulation and biohydogen fermentation are suggested. Moreover, fermentation effluent utilization such as for two-stage biogas production and further biohythane (a mixture of H₂ and CH₄) generation are also elucidated.     

Silage as source of bacteria and electrons for dark fermentative hydrogen production

In this study, grass silage was used both as a source of bacteria and as a substrate for dark fermentative hydrogen production. Silage is produced by lactic acid fermentation controlled by end point pH (<4.0). In this study, the fermentation of silage was successfully continued and directed to hydrogen production by neutralizing the pH. Highest hydrogen yield of 37.8 ± 5.8 mL H₂/g silage was obtained at 25 g/L of silage. The main soluble metabolites were acetate and butyrate with the final concentrations of 1.5 ± 0.2 and 0.5 ± 0.0 g/L, respectively. Bacteria present (at 25 g silage/L) included Ruminobacillus xylanolyticum, Acetanaerobacterium elongatum and Clostridium populeti and were involved in silage fermentation to hydrogen. In summary, this work demonstrates that grass silage becomes amenable to hydrogen fermentation by indigenous silage bacteria through pH neutralization.     

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.     

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.     

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 COD_init/(L day)). The highest hydrogen yield (HY) (44.73 ml/g COD_init) 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.     

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.     

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 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.     

Effects of pretreatment in a vortex layer apparatus on the properties of confectionery wastewater and its dark fermentation

Pretreatment prior to anaerobic digestion is an effective option for increasing the biodegradability of organic waste. Vortex layer apparatus (VLA) is considered one of the promising types of equipment for pretreatment. In this work, confectionery wastewater (CW) was pretreated in VLA for 1 and 3 min before dark fermentative hydrogen production in anaerobic upflow biofilters. The pretreatment resulted in a slight increase in soluble chemical oxygen demand (COD), soluble sugars and acetic acid, and a decrease in the concentration of propionic, butyric and caproic acids. Due to the abrasion of steel needles in VLA, the concentration of iron in the pretreated CW increased by 2.57 times. Hydraulic retention time in anaerobic upflow biofilters was gradually reduced from 5.6 to 1.8 and 1.3 days, which corresponded to organic loading rate of 2.0, 6.3 and 8.8 kg COD/(m³ day). Although the highest hydrogen yield (96.2 ± 8.1 ml/g COD) was obtained for non-pretreated CW, the pretreatment contributed to a significant increase in methane yield (39.2 ± 2.5 ml/g COD), possibly due to higher iron content (1.8 ± 0.3 mg/L). The highest energy production rate (4407 J/(L day)) was achieved after 3 min CW pretreatment. Thus, pretreatment in VLA can be a promising method for improving the biohythane production process.     

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.     

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.     

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.     

Lactate wastewater dark fermentation: The effect of temperature and initial pH on biohydrogen production and microbial community

Biohydrogen production using dark fermentation (hydrolysis and acidogenesis) is one of the ways to recover energy from lactate wastewater from the food-processing industry, which has high organic matter. Dark fermentation can be affected by the temperature, pH and the microbial community structure. This study investigated the effects of temperature and initial pH on the biohydrogen production and the microbial community from a lactate wastewater using dark fermentation. Biohydrogen production was successful only at lower temperature levels (35 and 45 °C) and initial pH 6.5, 7.5 and 8.5. The highest hydrogen yield (0.85 mol H₂/mol lactate consumed) was achieved at 45 °C and initial pH 8.5. The COD reduction achieved by fermenting the lactate wastewater at 35 °C ranged between 21 and 30% with the maximum COD reduction at pH 8.5, and at 45 °C, the COD reduction ranged between 12 and 21%, with the maximum at pH 7.5. At 35 °C, the lactate degradation ranged between 54 and 95%, while at 45 °C, it ranged between 77 and 99.8%. 16S rRNA sequencing revealed that at 35 °C, bacteria from the Clostridium genera were the most abundant at the end of the fermentation in the reactors that produced hydrogen, while at 45 °C Sporanaerobacter, Clostridium and Pseudomonas were the most abundant.     

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.     

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.