Biohydrogen production from cornstalk wastes by anaerobic fermentation with activated sludge

An anaerobic fermentation process to produce hydrogen from cornstalk wastes was systematically investigated in this work. Batch experiments numbered series I, II and III were designed to investigate the effects of acid pretreatment, enzymatic hydrolysis (enzymatic temperature, enzymatic time and enzymatic pH) on hydrogen production by using the natural sludge as inoculant. A maximum cumulative H₂ yield of 126.22 ml g⁻¹-CS (Cornstalk, or 146.94 ml g⁻¹-TS, Total Solid) and an average H₂ production rate of 9.58 ml g⁻¹-CS h⁻¹ were obtained from fermentation cornstalk with a concentration of 20 g/L and an initial pH of 7.0 at 36 °C through an optimal pretreatment process. The optimal process was that the substrate was soaked with an HCl concentration of 0.6 wt% at 90 °C for 2 h, and subsequently enzymatic hydrolysis for 72 h at 50 °C and pH 4.8 before fermentation. The biogas consisted of only H₂ and CO₂. In addition, the fermentation system was the typical ethanol-type fermentation according to ethanol and acetate as the main liquid by-products.     

Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Biohydrogen production from pretreated corn cobs

In this study, the co-fermentability of four different pretreated corn cob streams at different mixing ratios was assessed. The four streams, denoted DP, DS, HP, and HS, were: two dilute acid pretreatment comprising one purge and one squeeze and two high pressure autohydrolysis comprising one purge and one squeeze. The “Purge” stream was taken from the steam percolation reactor during cooling and the “Squeeze” stream was recovered from the cooked biomass with a pressing step. In addition, the impact of furfural and 5-hydroxymethylfurfural (HMF) on biohydrogen production potential was evaluated. The DP:DS mix at 50:50 by volume achieved the maximum H₂ yield of 265 (mL/gCOD sugars consumed). Furfural at concentrations of 0.21–1.09 g/L had no impact on H₂ production rates and yields and HMF was below the inhibitory threshold of 0.14 g/L. A positive correlation was observed between the monomeric-to-polymeric sugars ratio and H₂ production rates and yields.     

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.     

Biohydrogen production from biomass and industrial wastes by dark fermentation

Hydrogen is a clean energy carrier which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. This article summarizes the dark fermentative biohydrogen production from biomass. Types of potential biomass that could be the source for biohydrogen generation such as food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol are discussed in this article. Moreover, the microorganisms, factors affecting biohydrogen production such as undissociated acid, hydrogen partial pressure and metal ions are also discussed.     

Biohydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube

Hydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube (MWCNT-COOH) in repeated batch mode was studied. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) were employed to confirm immobilization of E. aerogenes successfully. The effect of MWCNT-COOH concentrations (0.2, 0.6, and 1.2 mg/mL) on hydrogen production was investigated. The present study showed that immobilized E. aerogenes on 1.2 mg/mL MWCNT-COOH resulted in higher hydrogen yield (2.2 moL/mol glucose), hydrogen production rate (2.72 L/L.h), and glucose degradation efficiency (96.20%) and shorter the lag phase (1 h) compared to the free E. aerogenes. Modified Gompertz and Logistic models were employed to predict the cumulative hydrogen production successfully.     

Biohydrogen production: Current perspectives and the way forward

Global research is moving forward in developing biological production of hydrogen (biohydrogen) as a renewable energy source to alleviate stresses due to carbon dioxide emissions and depleting fossil fuels resource. Biohydrogen has the potential to replace current hydrogen production technologies relying heavily on fossil fuels through electricity generation. While biohydrogen research is still immature, extensive work on laboratory- and pilot-scale systems with promising prospects has been reported. This work presents a review of advances in biohydrogen production focusing on production pathways, microbiology, as well as bioreactor configuration and operation. Challenges and prospects of biohydrogen production are also outlined.     

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.     

Net energy gain from solid organic wastes by dark fermentation and its end products

This study evaluated the feasibility of improving net energy gain from solid organic wastes by dark fermentation (DF) and its aqueous end products. Batch experiments were conducted with dairy cattle manure as a typical solid waste blended with sucrose at different sucrose:manure ratios. This study differs from previous DF studies in two aspects; these experiments were conducted at ambient temperature without any external nutrient supplements or pH control measures while previous studies had resorted to mesophilic conditions, nutrient supplements, and external pH control; this study evaluated the feasibility of DF in terms of net energy yield rather than in terms of hydrogen yield as in previous studies. Hydrogen yields (2.9–5.3 M H₂/M sucrose) and net energy gains (2.0–3.7 kJ/g COD) demonstrated in this study are higher than in previous reports. Based on this study, sucrose:manure ratio of 4.5% is suggested as the optimal ratio.     

Effect of pH shock on single-stage biohythane production using gel-entrapped anaerobic microorganisms

A novel single-reactor system having entrapped anaerobic microorganisms has been developed to co-produce H₂ and CH₄. pH is one of the key operating and environmental parameters affecting the performance of a bio-system. This work aimed to investigate the pH shock effects on the novel biohythane system. The experiments were suddenly changing the original cultivation pH value of 6 into 4, 5, 7 or 8 for 4 h. The results indicate that a short pH shock could be used to regulate H₂/CH₄ composition without notably affecting biogas yield and chemical oxygen demand (COD) removal. Peak biohythane production was obtained after the pH shock to 8, having H₂/CH₄ yields of 11.5 ± 1.6/44.8 ± 3.1 mL/g COD. During pseudo steady-state conditions of effective cultivation periods, the values of H₂ content in biohythane and COD removal efficiency were in ranges of 20–39% and 71–79%, respectively. The significances and applications of the experimental results have been discussed. The novelty of this work is elucidating a less-discussed field-operation problem of pH perturbances for a newly-developed biohythane system.     

Recent insights into consolidated bioprocessing for lignocellulosic biohydrogen production

Biohydrogen production via dark fermentation using fermentable sugars from biomass materials is a sustainable way of procuring biohydrogen. Lignocellulosic biomass is a potential renewable feedstock for dark fermentation, but its use is challenged by the recalcitrant nature and generation of certain fermentation inhibitors resulting in compromised fermentation performance. Consolidated bioprocessing (CBP), the successful integration of hydrolysis and fermentation of lignocellulosic biomass to desirable products, has received tremendous research attentions in recent years to boost renewable fuel production in an economically feasible way. A microbial strain capable of both biomass hydrolysis and hydrogen fermentation is critical for successful CBP-based hydrogen fermentation. This review provides comprehensive information on dark fermentation for hydrogen production using lignocellulosic biomass as a potential feedstock with a CBP approach. Consolidated bioprocessing of lignocellulosic biomass for biohydrogen production via native and recombinant microbial strains is discussed in detail. Potential bottlenecks in the above mentioned processes are critically analyzed and future research perspectives are presented.     

Comparative evaluation of the mesophilic and thermophilic biohydrogen production at optimized conditions using tequila vinasses as substrate

The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H₂) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 °C and 55 °C, and pH of 5.5 maximized the bio-H₂ production rates, 25.5 ± 0.01 NmL h⁻¹ and 169.9 ± 8.9 NmL h⁻¹ in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H₂ production rate of 519 ± 13 NmL-H₂ L⁻¹ d⁻¹ equivalent to 750 ± 19 NmL-H₂ L_vinasse⁻¹, while the mesophilic one 448 ± 23 NmL-H₂ L⁻¹ d⁻¹ and 647 ± 33 NmL-H₂ L_vinasse⁻¹, respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H₂ production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed.     

Biohydrogen production by dark fermentation: Experiences of continuous operation in large lab scale

Hydrogen is a clean energy carrier which can be used as fuel in fuel cells. Today, hydrogen is produced mainly by steam reforming of fossil fuels like natural gas or oil. But only hydrogen produced by renewable sources can be called clean energy production. One possibility for hydrogen production is the biological fermentation of biogenous wastes by hydrogen producing bacteria. For the experimental setup four 30-L-working-volume reactors were constructed for continuous biohydrogen production. As inoculum, heat-treated sludge of a wastewater treatment plant was used. Different hydraulic retention times (HRT) were tested and an organic loading rate (OLR) of 2–14 kg VS/m³*d. As starting substrate, waste sugar medium was used. The pH and other parameters were observed to find boundary conditions for a stable continuous process with a minimum of online-control measurements. The high concentration of organic acids in the reactor led to a very low pH, which was controlled manually and online > 4 up to 5.5, otherwise the biohydrogen production decreased rapidly. The gas amount varied with the different OLRs, but could be stabilised on a high level as well as the hydrogen concentration in the gas with 44–52%. No methane was detected in the gas. It turned out, that continuous biohydrogen production with stable gas amounts and qualities could be achieved at different operation conditions. The results showed, that the operation of a continuous biohydrogen reactor has to be observed very carefully to ensure a constant gas production, and that pH-control is necessary to ensure stable operation conditions.     

Biohydrogen production with lipid-extracted Dunaliella biomass and a new strain of hyper-thermophilic archaeon Thermococcus eurythermalis A501

Economic feasibility is important for the development of microalgae bioenergy industry. Dark fermentation of microalgal residue in a biorefinery context can improve the energy conversion efficiency of biomass and reduce the cost of microalgae industry. The present study proposes a promising dark fermentation model that combines thermophilic hydrogen-producing bacteria with algal residue substrates. Lipid-extracted Dunaliella residue can greatly improve hydrogen production by Thermococcus eurythermalis A501, the yields of which are more than four times higher than with algal cells as substrates. Under the optimal conditions of 2.5 g/L algal residue concentration and a 2:1 initial volume ratio of gas to liquid, the highest hydrogen yields of 192.35 and 183.02 mL/g volatile solid (VS) with algal residue of Dunaliella primolecta and D. tertiolecta are obtained, respectively, in less than 19 h without any pretreatment. This work may provide a biorefinery approach for comprehensive utilization of microalgae resources.     

Heat-shock treatment applied to inocula for H2 production decreases microbial diversities,interspecific interactions and performance using cellulose as substrate

The heat-shock pretreatment (HST) is a useful method to select for H₂-producing inocula when soluble substrates are employed. However, the HST has proven to have negative effects on the H₂ production performance from lignocellulosic substrates. We hypothesize that the negative effect of HST on H₂ production from lignocellulosic substrates is due to the loss of species involved in cellulose solubilization. In the present study, we tested this hypothesis by applying a heat-shock pretreatment (105 °C/24 h) on the microbial community for producing hydrogen from microcrystalline cellulose. Specifically, we compared a microbial community treated with 2-bromoethanesulfonate (BES-treated control) versus a heat-shock pretreated microbial community. For both experimental treatments, we determined the major fermentation products (hydrogen, acetic, butyric, propionic, and isovaleric acids), as well as the diversity of bacteria and fungi using Illumina MiSeq of amplicons in five sampling points. We found that HST immediately reduced alpha diversity of microbial communities, being fungi more affected than bacteria. We also found that the bacterial reduction in Comamonas, Ureibacillus, and Aneurinibacillus was related to a low hydrogen production in the heat-shock pretreated community. Strictly anaerobic fungi such as Orpinomyces, Cyllamyces, and Neocallimastix, which are recognized by their role in solubilization of fibrous materials, were unable to survive the HST. The reconstructed bacterial network predicted positive interactions between cellulase-producing and hydrogen-producing families. We conclude that the HST did not promote the high microbial diversity required for hydrogen production from cellulose.     

Biohydrogen production from paper industry wastes by SSF: A study of the influence of temperature/enzyme loading

The present work focused in assessing the hydrogen production from pretreated wastes of the paper industry (PIW) by simultaneous saccharification and fermentation (SSF) using anaerobic biofilms developed in natural fibers (ixtle) at different conditions. Anaerobic sludge from brewery wastewater treatment was used for biofilms and they were developed in plastic spheres covered with fiber cord from ixtle. The solid wastes of paper industry were previously pretreated with H₂SO₄ at 2.5% (v/v) at 120 °C by 30 min. The solids pretreated were hydrolyzed and fermented in batch reactors. All reactors were kept at an initial pH of 5.0 and three levels of enzyme loadings (10, 40 and 70 FPU/mL) and temperatures (35, 45, 55 °C) were assessed. The maximum hydrogen obtained (60.75 mmol/h*g volatile solids) was at 45 °C and 70 FPU/mL, moreover, no methane was detected in all cases.     

Effects of size and autoclavation of fruit and vegetable wastes on biohydrogen production by dark dry anaerobic fermentation under mesophilic condition

Fermentative hydrogen production from fruit and vegetable wastes (FVWs) through Dry Fermentation Technology (DFT) was studied through three independent experiments in order to find out the effect of particle size and autoclaving pretreatment on bio-hydrogen production from FVWs and as follows: (1) autoclaved FVWs with sizes < 5 cm (experiment I); (2) raw FVWs with sizes < 5 cm (experiment II) and (3) autoclaved FVWs with sizes > 5 cm (experiment III). The assay with autoclaved waste yielded a higher percentage of hydrogen in the headspace of the dry fermenter reaching a maximum value of 44% in experiment I. However, the maximum hydrogen production was obtained in experiment III with 14573 NmL at a yield of 23.53 NmL H₂/gVS. Profiling of the microbial communities by denaturing gradient gel electrophoresis (DGGE) indicated that the most prominent species were the genera Clostridium, Bifidobacterium, and Lactobacillus.     

Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).     

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.