Effect of fermentation conditions on biohydrogen production from lipid-rich food material

Among the basic components of organic materials, such as carbohydrate, protein, and lipid, the hydrogen yield of carbohydrate fermentation has been reported to be significantly higher than that of lipid. This study used lard as a model organic matter for lipid and investigated its H₂ production potential in batch anaerobic fermentation experiments under various combinations of stirring and CO₂-scavenging conditions. A significant increase in the hydrogen yield was observed in both CO₂-scavenging and stirring conditions; the CO₂-scavenging condition yield was 2.9 times higher than the stirring condition (116.7 and 40.3 mL H₂/g volatile solid [VS], respectively), which was much greater than reported previously. A maximal hydrogen yield of 185.8 mL H₂/g VS was obtained in the presence of both CO₂-scavenging and stirring, and the H₂ content of the total biogas was as high as 99% (v/v). In addition, there was less H₂ and more CH₄ production in the absence of CO₂-scavenging and/or stirring, which suggests that the consumption of H₂ and CO₂ for methanogenesis was the major mechanism of the poor hydrogen yield from lipid. The volatile fatty acids in all the tests consisted primarily of valeric (47.2–54.9%) and propionic acids (26.6–30.3%), and higher concentrations of these acids remained in the fermentation liquid without CO₂ removal. These results suggest that lipid-rich food waste is a potential source for H₂ production if the fermentation process is optimized to minimize the partial pressure of CO₂ and H₂ and restrain the activities of H₂-consuming bacteria.     

Modeling and monitoring of the acclimatization of conventional activated sludge to a biohydrogen producing culture by biokinetic control

The purpose of this work was to study the acclimatization of activated sludge as it evolved into an acidogenic culture capable of producing volatile fatty acids and hydrogen from glucose. The experiments were conducted in a Sequential Batch Reactor with a pH of 5, a temperature of 35 °C and in the absence of oxygen. Biomass, substrate and product concentrations were monitored during the experiment. The presence of aerobic, facultative and acidogenic microorganisms was determined for the initial seed, and their main kinetic and stoichiometric properties were determined. For the acidogenic microorganisms, μ_max, b and Y_x increased during the acclimatization process, reaching final values of 0.125 h⁻¹, 0.025 h⁻¹, 0.025 g mmol⁻¹, respectively. However, for the facultative microorganisms, μ_max and b decreased, whereas Y_x remained constant during the acclimatization process, reaching final values of 0.032 h⁻¹, 0.007 h⁻¹, 0.017 g mmol⁻¹, respectively. The evolution of the facultative organisms was significantly related to lactic acid production, whereas the evolution of the acidogenic microorganisms was related to hydrogen, acetic acid and butyric acid production. The biomass concentration of each group was estimated by modeling the experimental data.     

Electricity production by integration of acidogenic fermentation of fruit juice wastewater and fuel cells

In this work, the potential application of bio-hydrogen in a fuel cell was studied. To do that an activated sludge was acclimatized to an acidogenic culture producing bio-hydrogen. Once reached the steady state, several batch experiments were carried out by feeding a synthetic fruit juice industry wastewater to the acidogenic culture. The bio-hydrogen yield obtained when this synthetic fruit juice industry wastewater was fed was 1403 mol H₂/mol hexose and the hydrogen percent in gas phase was 57%. In an average size fruit juice industry, this bio-hydrogen yield corresponds to a bio-hydrogen production of about 6000 mol H₂/d. In the subsequent stage, a synthetic bio-hydrogen stream with the same composition of the cleaned bio-hydrogen obtained in the acidogenic fermentation was fed as fuel in a Fuel Cell, obtaining very similar power and polarization curves than that obtained when feeding pure hydrogen, differences lower than 10%. These results showed that the hydrogen stream obtained by the acidogenic fermentation could be used to produce electricity in a high temperature PEMFC.     

Simultaneous analysis of carbohydrates and volatile fatty acids by HPLC for monitoring fermentative biohydrogen production

The production of biohydrogen through anaerobic fermentation has received increasingly attention and has great potential as an alternative process for clean fuel production in the future. The monitoring of the stages of anaerobic fermentation provides relevant information about the bioprocess. The objective of this study is to propose a novel methodology for simultaneous analysis of sucrose, glucose, fructose and volatile fatty acids (VFAs), such as, acetic, propionic, isobutyric and butyric during anaerobic fermentation by using high-performance liquid chromatography (HPLC). The following chromatographic conditions were optimized: column Aminex HPX-87H, mobile phase consisting of H₂SO₄ 0.005 mol/L, flow rate of 1.0 mL/min and temperature of 55 °C. Sucrose, glucose and fructose were analyzed by refractive index detector (RI) while acetic, propionic, isobutyric and butyric acids were analyzed by ultraviolet (UV) detection at 210 nm. Some analytical parameters of validation, such as, linearity, selectivity, repeatability, intermediate precision, limit of detection and quantification, accuracy and robustness were evaluated. The proposed methodology was successfully applied in the determination of substrates and metabolites during different stages of biohydrogen production.     

Improvement of acidogenic fermentation using an acclimatized microbiome

Mixed fruit wastes (FW) are considered valuable organic wastes due to their polysaccharidic content. This study describes utilization of an effective acclimatized microbiome (AM) for enhanced conversion of FW into hydrogen and various value-added byproducts. Microbial acclimatization was used to accelerate two processes, hydrogenogenic acidogenesis and carboxylic chain elongation, which simultaneously produced hydrogen and C4C7 carboxylates. AM showed 77 mL g‾¹ VS of hydrogen yield with 31% higher specific hydrogen production potential (SHPP) compared to 55 mL g‾¹ VS with an unacclimatized microbiome (UM). Production of carboxylates was also 19% higher in the AM. Taxonomic analysis of the microbiome revealed the microbial shift to Firmicutes as the most dominant phylum (99%). Clostridium, Hydrogenoanaerobacterium, Paraclostridium, Anaerosalibacter, Tissierella, and Tepidanaerobacter were preeminent genera in the AM, confirming their predominant role in dual processes. Thus, utilization of an AM enhanced the hydrogenogenic acidogenic fermentation of FW with simultaneous carboxylic chain elongation, yielding high-value products.     

Comparison of thermophilic and hyperthermophilic dark fermentation with subsequent mesophilic methanogenesis in expanded granular sludge bed reactors

Herein, dark fermentation (DF, V = 5.5 L) and subsequent mesophilic methanogenesis (V = 43.5 L) are run as expanded granular sludge bed reactors (EGSB) at thermophilic (υDF = 60 °C) and hyperthermophilic (υDF = 80 °C) temperatures. A synthetic glucose wastewater is run with a 22.5 g/L chemical oxygen demand (COD) and 48–9 h hydraulic retention times (HRTs), giving organic loading rates (OLRs) of 11–60 g COD/L/d for DF. The maximum hydrogen production rate (HPR) is HPR = 3.0 m³/m³/d for HRT = 9 h with a 50 L/kg COD hydrogen yield (HY) and 40 vol% H₂. Methane production rate (MPR) reaches MPR = 2.6 m³/m³/d with 70 vol% CH₄ at HRT = 2.8 d. The highest H₂ yields are HY = 180 L/kg COD with 53 vol% H₂ (thermophilic, HRT = 48 h). Hyperthermophilic temperatures led to lower HPRs (0.7 m³/m³/d) and MPRs (1.6 m³/m³/d). 53% of Thermoanaerobacterium thermosaccharolyticum as an H₂ producer are found. Discoloration of granular sludge from black to white and granule stability was observed in DF.     

Metabolic shift and electron discharge pattern of anaerobic consortia as a function of pretreatment method applied during fermentative hydrogen production

We have made an attempt to evaluate the variation in the electron discharge (ED) pattern of anaerobic consortia as a function of pretreatment viz., chemical, heat-shock, acid and oxygen-shock in comparison with untreated mixed consortia during fermentative hydrogen (H₂) production. Experiments were performed with dairy wastewater as substrate using anaerobic mixed consortia as biocatalyst (pretreated individually and in combination). Cyclic voltammetry (CV) elucidated significant variation in the ED pattern of mixed consortia along with H₂ production and substrate degradation (SD) as a function of pretreatment method applied. Higher ED was observed with all pretreated consortia which can be attributed to the stable proton (H⁺) shuttling due to the suppression of methanogenic activity. Oxygen-shock method and untreated consortia showed lower H₂ production and higher SD among the variations studied, while, combined pretreated consortia resulted higher H₂ production and lower SD. Lower ED observed with untreated consortia suggests the H⁺ reduction during methanogenesis rather than the inter-conversion of metabolites, which is presumed to be necessary for H₂ production. ED observed with combined pretreated consortia corroborated well with the observed H₂ production. Redox pairs were visualized on the voltammograms with almost all the experimental variations studied except untreated consortia. The potentials (E₀) of redox pairs observed were corresponding to intracellular electron carriers viz., NAD⁺/NADH (E₀ −0.32 V) and FAD⁺/FADH₂ (E₀ −0.24 V).     

Upscaling of biohydrogen production process in semi-pilot scale biofilm reactor: Evaluation with food waste at variable organic loads

The present account focuses on upscaling of biohydrogen (H₂) production at semi-pilot scale bioreactor using composite food waste. Experiments were conducted at different organic load (6, 12, 18, 30, 40, 50 and 66 g COD/l) conditions. H₂ production increased with an increasing organic load up to 50 g COD/l (9.67 l/h) followed by 40 g COD/l (6.48 l/h), 30 g COD/l (1.97 l/h), 18 g COD/l (0.90 l/h), 12 g COD/l (0.78 l/h) and 6 g COD/l (0.32 l/h). H₂ production was affected by acidification (pH drop to 3.96) at 66 g COD/l operation due to the excess accumulation of soluble metabolites (5696 mg VFA/l). Variation in organic load of food waste influenced the overall hydrogen production efficiency.     

Enhancing photo fermentative hydrogen production using ethanol rich dark fermentation effluents

The present study demonstrates the feasibility of a two-phase biorefinery process applied to waste substrates producing ethanol rich effluents. The process includes a dark fermentation step followed by photo fermentation and it is able to optimize hydrogen production from waste biomass. The study was conducted using winery wastewater as feedstock. The results indicate that no additional treatments are required when an appropriate dilution of the initial waste is applied. Microbial consortia contained in the winery wastewater promoted a fermentative ethanol pathway. The ethanol rich effluent was converted into hydrogen by phototrophic microorganisms. Despite the presence of inhibiting compounds, the adoption of a mixed phototrophic culture allowed to obtain good results in terms of hydrogen production. Specifically, up to 310 mLH₂ gCOD_consumed^?1 were obtained in the photo fermentative stage. The effectiveness of ethanol rich dark fermentation effluents for hydrogen production enhancement was demonstrated. Noteworthy, polyhydroxybutyrate was also produced during the experiments. The work faces two of the major challenges in the sequential dark fermentation and photo fermentation technology applied to real waste substrates: the minimization of pre-treatments and the enhancement of the hydrogen production yields using ethanol rich DFEs.     

Evaluation of sludge liquors from acidogenic fermentation and thermal hydrolysis process as feedstock for microbial electrolysis cells

In this study, mesophilic acidogenic fermentation, thermophilic acidogenic fermentation, and thermal hydrolysis process (THP) were compared to generate sludge liquors for bioenergy recovery with microbial electrolysis cells (MECs). The results showed that THP at 170 °C was the most effective for hydrolysis of particulate organics in sewage sludge, while fermentation under thermophilic temperature led to the highest accumulation of volatile fatty acids (VFAs) in sludge liquor. However, THP yielded the highest percentage of acetate in VFAs, which resulted in superior MEC performance compared to fermented sludge liquors in terms of current density (2.7 vs. ~1.3 A/m²), coulombic efficiency (50% vs. 31–34%), bio-H₂ potential (1114 vs. 839–881 mL), and H₂ production rate (50.3 mL/d vs. 28–32 mL/d). The utilization sequence of the VFAs was found to be acetate > butyrate > propionate. Overall, our results show that generating sludge liquors through THP could provide a feasible solution to produce bio-H₂ from sewage sludge; however, coulombic efficiencies should be further improved before practical application.     

High hydrogen production rate in a submerged membrane anaerobic bioreactor

Batch cultivation of an anaerobic consortium fed with glucose as sole carbon source showed a sharp decrease of the hydrogen productivity when volatile fatty acids (VFA) concentration exceeded 12.5 g L^?1. To avoid VFA accumulation, fermentative batch cultures were thereafter carried out with a submerged membrane anaerobic bioreactor to continuously remove hydrogen fermentation co-products, while retaining the biomass. The membrane made it possible to separate the residence times of bacterial biomass and hydraulic part. With this technology, average and maximal productivities reached 0.75 and 2.46 L_H2 L^?1 h^?1, corresponding to an increase of 44 and 51% in comparison to the control, respectively. By removing the VFAs from the cultivation medium, H₂-producing pathways were favored, confirming the metabolic inhibitory effects of co-product accumulation in fermentation medium. Such hydrogen productivity is one of the highest values encountered in the literature. Readily implementable, such technology offers a good opportunity for further developing high rate hydrogen fermentation bioprocesses.     

Influence of pH, temperature and volatile fatty acids on hydrogen production by acidogenic fermentation

The aim of this work was to study the influence of pH and temperature on acidogenic fermentation and bio-hydrogen production. A centered factorial design was generated with respect to pH (4-6 units) and temperature (26-40 °C), and these conditions were used in batch experiments. Biomass cultivation was conducted in a sequential batch reactor (SBR). A mixed-acidogenic culture enriched from activated sludge and fed with a 9 g/l glucose solution was used in the experiments. At low pH values, hydrogen production was favored when the temperatures were low, a result contrary to those described in literature. Working at higher temperatures reduced the length of the lag phase. Additionally, the hydrogen production rate was increased at these temperatures. These opposite trends indicated that an inhibition effect occurred during the experiment. Hydrogen production was studied by using a response surface methodology, being the highest hydrogen production occurred at pH 5.4 and 26 °C. Regarding to the relationship between the hydrogen and acid production, the hydrogen produced per unit of acetate produced increased as the pH increased. On the other hand, hydrogen produced from other acids was constant and similar to theoretical yields. These values of hydrogen produced per unit of acid produced allowed to estimate the experimental hydrogen production. This result indicated that pH was the most important factor in acidogenic fermentation.     

Biotechnological approach to generate green biohydrogen through the utilization of succinate-rich fermentation wastewater

Photofermentation seems to be an attractive mode of generating biohydrogen from fermentation effluent. Use of succinate fermentation effluent, however, has not been reported. Rhodobacter sphaeroides KKU-PS1 and Rhodopseudomonas palustris were acclimatised in succinate. It was determined that the KKU-PS1 was superior with respect to hydrogen productivity and was selected for further experiments. Photofermentation in succinate by the KKU-PS1 validated, generating 1217 mL H₂/L of cumulative hydrogen at a maximum rate of 6.7 mL H₂/L/h. Photofermentation from each single carbon sources that are components of effluent was performed and it was determined that acetate and succinate promoted the fastest growth of KKU-PS1 and hydrogen evolution, respectively. Photofermentation by the strain using mixed substrates mimicking diluted bio-succinate effluent produced yielded 1005 mL H₂/L cumulative hydrogen at a maximum rate of 4.1 mL H₂/L/h. The study highlighted potential of utilizing bio-succinate fermentation effluent for biohydrogen production, with further optimization required.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

Strong pH dependence of hydrogen production from glucose by Rhodobacter sphaeroides

Purple non-sulfur bacteria (PNSB) are well known for converting short-chain organic acids to H₂, however, a decrease in pH caused by metabolic acids production limited H₂ production during the photo-fermentation from glucose. Here we address why volatile fatty acids (VFA) excreted as fermentation products cannot be further degraded by R. sphaeroides that readily use them. We found that the photo-fermentation with pH controlled at 6.9 ± 0.1 resulted in a 90% increase of H₂ yield and a 107.6% increase in volume H₂ production relative to the pH-uncontrolled culture. Comparative fermentations on glucose at pH 5.8 and pH 7.1 using culture medium supplemented with 50% spent fermentation broth demonstrated that low pH alone is not the limiting factor and compounds present in the supernatants along with pH decrease were the most inhibitory to H₂ production. The impact of byproducts VFA on phototrophic H₂ production was dependent on both the pH and VFA concentrations; even 7 mM VFA addition totally inhibited H₂ production from glucose at pH 5.4. H₂ production with pH control for the Δhup strain was not discernibly different from the parent strain, which are all significantly higher than high-performance strains by metabolic engineering. These results demonstrate that pH dependent VFA inhibition can be turned into a driving force for enhanced H₂ production from glucose by pH regulation.     

Hydrogen gas production by electrohydrolysis of volatile fatty acid (VFA) containing dark fermentation effluent

Dark fermentation effluents of wheat powder (WP) solution containing different concentrations of volatile fatty acids (VFAs) were subjected to low voltage (1–3 V) DC current to produce hydrogen gas. Graphite and copper electrodes were tested and the copper electrode was found to be more effective due to higher electrical conductivity. The effects of solution pH (2–7), applied voltage (1–3 V) and the total VFA (TVFA) concentration (1–5 g L⁻¹) on hydrogen gas production were investigated. Hydrogen production increased with decreasing pH and became maximum at pH = 2. Increases in applied voltage and the TVFA concentration also increased the cumulative hydrogen formation. The most suitable conditions for the highest cumulative hydrogen production was pH = 2, with 3 V applied voltage and 5 g TVFA L⁻¹. Up to 110 ml hydrogen gas was obtained with 5 g L⁻¹ TVFA at pH = 5.8 and 2 V applied voltage within 37.5 h. The highest energy efficiency (56%) was obtained with the 2 V applied voltage and 10.85 g L⁻¹ TVFA. Hydrogen production by electrolysis of water in control experiments was negligible for pH > 4. Hydrogen production by electrohydrolysis of VFA containing anaerobic treatment effluents was found to be an effective method with high energy efficiency.     

Effect of pre-treatment and hydraulic retention time on biohydrogen production from organic wastes

This study investigated the effect of pre-treatment and hydraulic retention time (HRT) on biohydrogen production from organic wastes. Various pre-treatments including thermal, base, acid, ultrasonication, and hydrogen peroxide were applied alone or in combination to enhance biohydrogen production from potato and bean wastewater in batch tests. All the pre-treated samples showed higher hydrogen production than the control tests. Hydrogen peroxide pre-treatment achieved the best results of 939.7 and 470 mL for potato and bean wastewater, respectively. Continuous biohydrogen production from sucrose, potato and bean wastewater was significantly influenced by reducing the HRT as 24, 18 and 12 h. Sucrose and potato showed similar behavior, where the hydrogen production rate (HPR) increased with decreasing the HRT. Optimum hydrogen yield results of 320 mL-H₂/g-VS (sucrose) and 150 mL-H₂/g-VS (potato) were achieved at HRT of 18 h. Bean wastewater showed optimum HPR of 0.65 L/L.d with hydrogen yield of 80 mL-H₂/g-VS at 24 h HRT.     

Different role of focA and focB encoding formate channels for hydrogen production by Escherichia coli during glucose or glycerol fermentation

The dependence of H₂ production on the formate channels, FocA and FocB, by Escherichia coli at pH 5.5, 6.5 and 7.5 was shown using focA and focB mutants and comparing with the wild type. Moreover, effect of exogenous addition of formate (10 mM) on H₂ production was allotted. The results acquired propose that during glucose fermentation formate import can occur through FocB at different pHs; external formate drives FocA to import direction. However, during glycerol fermentation formate might be imported through FocB, whereas formate is exported preferentially through FocA at pH 7.5.     

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.     

Operational behavior of a hydrogen extractive membrane bioreactor (HEMB) during mixed culture acidogenic fermentation

Fermentative hydrogen production requires a continuous products-removal and effective upgrading steps to improve its general performance. Therefore, implementation of new technologies capable of achieving both requirements is essential. We present the operational behavior of a new process concept based on integration of membranes for gas separation and fermentation technology. This process, which we term as hydrogen extractive membrane bioreactor consists of coupling two dense polymeric membranes to a hydrogen producing culture. The process automatization of this system was essential to maintain the proper operational pressures in the membrane module and in the bioreactor-gas-phase. This system was able to extract and partially separate the hydrogen and carbon dioxide generated. The hydrogen partial pressure was reduced from 55.5 to 49 KPa, which means an increase of hydrogen yield of 16.3% (1.1–1.28 mol-H₂/mol-glucose). Simultaneously, the implemented system generated a final hydrogen stream 13% (v/v) more concentrated than a conventional process.