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.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

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.     

Promoting dark fermentation for biohydrogen production: Potential roles of iron-based additives

Dark fermentation (DF) is a promising technology for biohydrogen production. Low efficiency of biohydrogen production is a bottleneck of the scale-up prospects for DF. Additives have been extensively studied to improve the biohydrogen production efficiency. Among of them, iron-based additives present a promising application potential due to their demonstrated significant enhancement of DF efficiency and among the low-cost bioactive agents. However, current reviews mainly examined the effects of nano-materials on DF and an in-depth analysis of enhancing mechanisms with addition of iron-based additives in DF is still lacking. To this end, this article comprehensively reviewed and evaluated the effects of iron-based additives on DF. Further, the potential mechanisms, including altering metabolic pathways, improving activities of microbes and enzymes, promoting electron delivery, and enriching hydrogen-producing bacteria, were discussed. Lastly, prospects and challenges of iron-based additives for subsequent research and large-scale application for DF were summarized.     

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.     

Application of nanotechnology in dark fermentation for enhanced biohydrogen production using inorganic nanoparticles

Fermentation is an important innovation by mankind and this process is used for converting organic substrate into useful products. Using natural conditions, specifically, light and dark conditions, photo-fermentation and dark fermentation techniques can be developed and operated under controlled conditions. Generally, products such as biofuels, bioactive compounds and enzymes have been produced using the dark fermentation method. However, the major requirement for today's industralized world is biofuels in its clean and pure forms. Biohydrogen is the most efficient and cleanest form of energy produced using dark fermentation of organic substrates. Nevertheless, the quantity of biohydrogen produced via dark fermentation is low. In order to increase the product quantity and quality, several internal and external stress or alterations are made to conventional fermentation conditions. In recent times, nanotechnology has been introduced to enhance the rate of dark fermentation. Nanoparticles (NPs), specifically, inorganic NPs such as silver, iron, titanium oxide and nickel have increased the production rate of biohydrogen. Therefore, the present review focuses on exploring the potential of nanotechnology in the dark fermentation of biohydrogen production, the mechanisms involved, substrates used and changes to be made to increase the production efficiency of dark fermentation.     

Deoiled algal cake as feedstock for dark fermentative biohydrogen production: An integrated biorefinery approach

An integrated biorefinery approach utilizing deoiled algal cake (after lipid extraction) as potential feed-stock for biohydrogen (H₂) production using selectively enriched acidogenic consortia as biocatalyst was evaluated. Algae pretreated extract (AP-E) documented maximum H₂ production rate (HPR), cumulative H₂ production (CHP) and specific H₂ yield (SHY) with higher substrate degradation (65%) in terms of COD removal efficiency than other conditions, which is a good sign for waste remediation. Along with the biohydrogen production and substrate removal the consortia also produced good amount of volatile fatty acids (VFA). VFA production in fermentation media resulted in reactor pH drop. The study depicted the feasible use of deoiled algal biomass as feed-stock for H₂ production in the framework of biorefinery.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.     

Modeling dark fermentation for biohydrogen production: ADM1-based model vs. Gompertz model

Biohydrogen production by dark fermentation in batch reactors was modeled using the Gompertz equation and a model based on Anaerobic Digestion Model (ADM1). The ADM1 framework, which has been well accepted for modeling methane production by anaerobic digestion, was modified in this study for modeling hydrogen production. Experimental hydrogen production data from eight reactor configurations varying in pressure conditions, temperature, type and concentration of substrate, inocula source, and stirring conditions were used to evaluate the predictive abilities of the two modeling approaches. Although the quality of fit between the measured and fitted hydrogen evolution by the Gompertz equation was high in all the eight reactor configurations with r² ∼0.98, each configuration required a different set of model parameters, negating its utility as a general approach to predict hydrogen evolution. On the other hand, the ADM1-based model (ADM1BM) with predefined parameters was able to predict COD, cumulative hydrogen production, as well as volatile fatty acids production, albeit at a slightly lower quality of fit. Agreement between the experimental temporal hydrogen evolution data and the ADM1BM predictions was statistically significant with r² > 0.91 and p-value <1E-04. Sensitivity analysis of the validated model revealed that hydrogen production was sensitive to only six parameters in the ADM1BM.     

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.     

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.     

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.     

Optimized model of fermentable sugar production from Napier grass for biohydrogen generation via dark fermentation

Biohydrogen is a fossil-fuel alternative. Lignocellulosic biomass is a complex part of cellulose-to-simple sugar production. Napier grass, one of the lignocellulosic biomasses, is best for biofuels or biochemicals. The dark fermentation process of Napier grass for biohydrogen proved both cost-effective and environmentally friendly. This grass contains cellulose, hemicellulose and lignin were 35.44 ± 2.01, 20.05 ± 1.55, and 28.473 ± 1.34, respectively. Sodium hydroxide was used in different concentrations to delignify lignocellulose and improve grass glucose recovery. Fermentative hydrogen production from grass biomass processing by microflora was optimized in terms of pH (4.5–7.0) and mesophilic condition (35 ± 2 °C). In this study, mesophilic conditions favored maximum hydrogen production (763.34 ml), indicating that pH 5.5 was suitable for dark-fermentative hydrogen production; study results showed Napier grass could be used successfully for dark fermentation to produce biohydrogen.     

Impact of pretreatment on food waste for biohydrogen production: A review

Food waste (FW) can be utilized as a raw material to produce energy such as hydrogen via fermentation, which is a more attractive and environmentally friendly approach compared to incineration and land-filling. Food waste must be pretreated before being used in various biological processes. The choice of the pretreatment method usually depends on the composition of the food waste. Therefore, various pretreatment methods generally employed to treat FW, including physical, physiochemical, chemical and biological pretreatments, are summarized in this review. The different pretreatment methods are compared in terms of their efficiency and biohydrogen yield. Additionally, the energy efficiencies of the various pretreatment methods are compared, thereby leading to the selection of the most efficient pretreatment method.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h^?1), a higher butyric acid production of 6.8 g·L^?1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h^?1, with a constant hydrogen production of 507 mL·h^?1, corresponding to a volumetric rate of 6.10 L·L^?1 d^?1, and a yield of 2.39 mol of H₂ per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.     

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.     

Development of a method for biohydrogen production from wheat straw by dark fermentation

Lignocellulosic biomass contains approximately 70-80% carbohydrates. If properly hydrolyzed, these carbohydrates can serve as an ideal feedstock for fermentative hydrogen production. In this research, batch tests of biohydrogen production from acid-pretreated wheat straw were conducted to analyze the effects of various associated bioprocesses. The objective of the pretreatment phase was to investigate the effects of various sulfuric acid pretreatments on the conversion of wheat straw to biohydrogen. When sulfuric acid-pretreated solids at a concentration of 2% (w/v) were placed in an oven for 90 min at 120 °C, they degraded substantially to fermentative gas. Therefore, wheat straw that is pre-treated under the evaluated conditions is suitable for hydrolysis and fermentation in a batch test apparatus. Five different conditions were evaluated in the tests, which were conducted in accordance with standard batch test procedures (DIN 38414 S8): fresh straw, pre-treated straw, supernatants derived from acid hydrolyzation, Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF). The SSF method proved to be the most effective and economical way to convert wheat straw to biohydrogen. The hydrogen yield by this method was 1 mol H₂/mol glucose, which resulted from 5% carbon degradation (η_{C, gas}) or the equivalent of 64% of the hydrogen volume that was produced in the reference test (glucose equivalent test). This method also proved to have the shortest lag phase for gas production. The supernatants derived from acid hydrolysis were very promising substances for continuous tests and presented excellent characteristics for the mass production of biohydrogen. For example, a 1.19 mol H₂/mol glucose (76% glucose equivalent) yield was achieved along with a 52% carbon degradation.     

Multiscale mixing analysis and modeling of biohydrogen production by dark fermentation

Hydrogen production by dark fermentation (DF) from wastewater, food waste, and agro-industrial waste combines the advantages to be renewable, sustainable and environmentally friendly. But this attractive process involves a three-phase gas-liquid-solid system highly sensitive to mixing conditions. However, mixing is usually disregarded in the conventional strategies for enhancing biohydrogen productivity, even though H₂ production can be doubled, e.g. versus of reactor design (0.6–1.5 mol H₂/mol hexose). The objective of this review paper is, therefore, to highlight the key effects of mixing on biohydrogen production among the abiotic parameters of DF. First, the pros and cons of the different modes of mixing in anaerobic digesters are described. Then, the influence of mixing on DF is discussed using recent data from the literature and theoretical analysis, focusing on the multiphase and multiscale aspects of DF. The methods and tools available to quantify experimentally the role of mixing both at the local and global scales are summarized. The 0-D to 3-D strategies able to implement mixing in fermentation modeling and scale-up procedures are examined. Finally, the perspectives in terms of process intensification and scale-up tools using mixing optimization are discussed with the issues that are still to be solved.     

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.