Prolongation of H2 production during mixed carbon sources fermentation in E. coli batch cultures: New findings and role of different hydrogenases

Hydrogen (H₂) gas production in batch cultures was studied upon utilization of the mixture of glucose, glycerol and formic acid by Escherichia coli BW25113 wild type (wt) at pH of 5.5–7.5. At pH 7.5H₂ was continuously produced during 240 h but at pH 6.5 and 5.5 it was detected till 168 h and 120 h, respectively. Specific growth rate (μ) of wt was the highest (1.05 h^?1) at pH 6.5. Moreover, at pH 5.5 in hycE μ decreased by ～4.14 fold compared to wt, suggesting major role of Hyd-3 in cell growth. H₂ yield (8.8 mmol H₂ L^?1) was the highest at pH 7.5. In hybC H₂ yield was increased ～1.62 fold than in wt. These data might be applied for biomass and biohydrogen production from various organic wastes where mixtures of carbon sources are present.     

Pentoses,hexoses and glycerin as substrates for biohydrogen production: An approach for Brazilian biofuel integration

Biofuels production in Brazil is a traditional activity, and it has becoming more important each year. Within this context, biohydrogen production could exploit residual streams from first generation ethanol (ethanol 1G), second generation ethanol (ethanol 2G) and biodiesel production. Therefore hexoses, pentoses and glycerin were tested as substrates for hydrogen production. Firstly, the effects of different inoculum pretreatments (acid, alkaline and heat) on bacterial communities' performance were evaluated through the levels of Clostridium hydrogenase expression. The heat pretreated inoculum provided the highest yield of H₂ (4.62 mol H₂/mol sucrose) and also the highest level of hydrogenase expression, 64 times higher when compared with untreated inoculum after 72 h. Then C5 and C6 sugars and also glycerin were tested for H₂ production (35 °C and pH 5.5), which resulted in promising yields of H₂: sucrose (4.24 mol H₂/mol sucrose), glucose (2.19 mol H₂/mol glucose), fructose (2.09 mol H₂/mol fructose), xylose (1.88 mol H₂/mol xylose) and glycerin (0.80 mol H₂/mol glycerin).     

Coupling dark fermentation and microbial electrolysis to enhance bio-hydrogen production from agro-industrial wastewaters and by-products in a bio-refinery framework

The aim of this work is to evaluate biohydrogen production from agro-industrial wastewaters and by-products, by combining dark fermentation and microbial electrolysis in a two-step cascade process. Such coupling of both technologies constitutes a technological building block within a concept of environmental biorefinery where sustainable production of renewable energy is expected.Six different wastewaters and industrial by-products coming from cheese, fruit juice, paper, sugar, fruit processing and spirits factories were evaluated for the feasibility of hydrogen production in a two-step process. The overall hydrogen production when coupling dark fermentation and microbial electrolysis was increased up to 13 times when compared to fermentation alone, achieving a maximum overall hydrogen yield of 1608.6 ± 266.2 mLH₂/gCOD_consumed and a maximum of 78.5 ± 5.7% of COD removal.These results show that dark fermentation coupled with microbial electrolysis is a highly promising option to maximize the conversion of agro-industrial wastewaters and by-products into bio-hydrogen.     

New insights into the systems for heterologous synthesis and maturation of hydrogenases,the most efficient biohydrogen producers

Biohydrogen is considered as an important key to a sustainable world power supply and is currently being seen as the versatile fuel of the future, with the potential to replace fossil fuels. The most efficient biohydrogen producers are hydrogenases. Nevertheless, due to the complex maturation processes of these enzymes, their heterologous production leaves some intriguing points not elucidated up to now. The limit of our understanding in this field makes a barrier for hydrogenases application in a variety of biotechnological processes. This review focuses on recent progresses in the development of heterologous production systems and cell-free maturation systems for the biosynthesis of active [Fe–Fe] and [Ni–Fe] hydrogenases. It also highlights some up to now un-discussed questions on the probable existence of unknown machinery able to maturate [Fe–Fe] hydrogenases or a contribution of the [Ni–Fe] hydrogenases maturases to the formation of an active H-cluster for the [Fe–Fe] hydrogenases.     

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.     

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.     

Glucose electro-fermentation with mixed cultures: A key role of the Clostridiaceae family

Electro-fermentation is a new type of bioprocess combining the concepts of fermentation and electro-microbiology to improve the conversion of organic substrates into valuable fermentation products. During electro-fermentation metabolic profiles could be redirected by the presence of polarized electrodes through changes in the microbial communities in the dark fermentation. This paper aims to investigate the influence of the bacterial community composition on glucose electro-fermentation in batch electro-systems. Our results showed that the initial microbial community significantly impacted the final microbial community and related metabolic patterns. During electro-fermentation, the H₂ yield was increased using anaerobic sludge but decreased using activated sludge as inocula. While using other inocula from similar origins, no differences between electro-fermentation and traditional fermentation were evidenced. The relative abundance of Clostridiaceae family members in the inoculum appeared to be a determining factor affecting the global performances. These findings provide new insights on electro-fermentation mecanisms occurring in mixed cultures.     

Modeling and simulation of net energy gain by dark fermentation

For dark fermentation (DF) to be accepted as a sustainable process for biohydrogen production, the net energy gain should be positive and as high as possible. A theoretical approach is proposed in this study to evaluate the net energy gain possible from hydrogen generated by the DF process as well as from the end products of DF via anaerobic digestion (AD) and microbial fuel cells (MFC). Experimental data on hydrogen evolution and aqueous end products formation from sucrose and from sucrose/dairy manure blends were used to validate the proposed approach for estimating net energy gain via DF, DF + AD, DF + MFC. Good agreement was found between the experimental and predicted net energy gain values, with overall correlation coefficient of 0.998. Based on the results of this study, DF + MFC is recommended as the best combination to maximize net energy gain.     

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.     

Bio-hydrogen production from cheese whey powder (CWP) solution: Comparison of thermophilic and mesophilic dark fermentations

Cheese whey powder (CWP) solution was used as the raw material for hydrogen gas production by mesophilic (35 °C) and thermophilic (55 °C) dark fermentations at constant initial total sugar and bacteria concentrations. Thermophilic fermentation yielded higher cumulative hydrogen formation (CHF = 171 mL), higher hydrogen yield (111 mL H₂ g⁻¹ total sugar), and higher hydrogen formation rate (3.46 mL H₂ L⁻¹ h⁻¹) as compared to mesophilic fermentation. CHF in both cases were correlated with the Gompertz equation and the constants were determined. Despite the longer lag phase, thermophilic fermentation yielded higher specific H₂ formation rate (2.10 mL H₂ g⁻¹cells h⁻¹). Favorable results obtained in thermophilic fermentation were probably due to elimination of H₂ consuming bacteria at high temperatures and selection of fast hydrogen gas producers.     

Optimization of biohydrogen inoculum development via a hybrid pH and microwave treatment technique – Semi pilot scale production assessment

The interactive effect of a hybrid pH and microwave pre-treatment on a mixed inoculum for biohydrogen production was investigated. Response surface methodology (RSM) was employed to obtain the optimum pre-treatment conditions of pH, microwave duration and microwave intensity for maximum hydrogen yield. The obtained model had a coefficient of correlation (R²) of 0.87. The optimum inoculum pre-treatment conditions predicted were pH 11 and 2 min microwave treatment at 860 W and the validation experiments demonstrated a 32.41% increase on hydrogen yield.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.