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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Kinetic analysis of biohydrogen production from complex dairy wastewater under optimized condition

Present work describes a kinetic analysis of various aspects of biohydrogen production in batch test using optimized conditions obtained previously. Monod model and Logistic equation have been used to find growth kinetic parameters in batch test under uncontrolled pH. The values of μ_m, K_s, and X_m were 0.64 h⁻¹, 15.89 g-COD L⁻¹, and 7.26 g-VSS L⁻¹, respectively. Modified Leudeking-Piret and Michaelis–Menten equation corroborates a flux of energy to hydrogen production pathway and energy sufficiency in the system. Modified Gompertz equation illustrates that the overall rate and hydrogen yield at 15 g-COD L⁻¹ was higher compared to a dark fermentation of other wastewaters. Besides, Andrew's equation also suggests that since the higher value of K_I (19.95 g-COD L⁻¹), k (255 mL h⁻¹ L⁻¹) was not inhibited at high S. The experimental results implied that the entire products during the fermentation process were growth and substrate degradation associated. The result also confirms that the acetate and butyrate were substantially used for hydrogen production in acidogenic metabolism under uncontrolled pH.     

Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

An integrated biological hydrogen production process based on ethanol-type fermentation and bipolar membrane electrodialysis

An integrated bio-hydrogen production system involving fermentative hydrogen production and product separation is proposed. In this process, microorganisms conduct ethanol-type fermentation and generate H₂ gas in anaerobic bioreactor, and acetate is removed from fermentation broth by using a two chamber bipolar membrane electrodialysis as separation unit. A comparative study of fermentative hydrogen production of Ethanoligenens harbinese B49 in the integrated system with traditional fermentation process was carried out. Compared to traditional process, accumulated H₂ elevated 23%, glucose utilization ratio increased by 135% and cell growth increased by 27% in the integrated system. The specific hydrogen production rate reached 2.2 mol H₂/mol glucose, indicating that separation of acetate from fermentation system has a great role in promoting hydrogen producing capacity. Bipolar membrane electrodialysis showed high acetate separation efficiency and low glucose loss rate. In the integrated system, pH could be used to direct electrodialysis operation, since it has an exponential correlation with acetate concentration in fermentation broth. These results provide a new method for achieving efficient and stable H₂ production with simultaneous glucose recovery and acetate inhibition release.     

Fermentative biohydrogen production from starch-containing textile wastewater

The present study deals with the biohydrogen production from starch-containing wastewater collected from the textile industry in Taiwan. The effects of inoculums collected from different sources (sewage sludge, soil and cow dung), substrate concentrations (5–25 g COD/L) and pH (4.0–8.0) on hydrogen production from wastewater were investigated.     

Integration of acidogenic and methanogenic processes for simultaneous production of biohydrogen and methane from wastewater treatment

Feasibility of integrating acidogenic and methanogenic processes for simultaneous production of biohydrogen (H₂) and methane (CH₄) was studied in two separate biofilm reactors from wastewater treatment. Acidogenic bioreactor (acidogenic sequencing batch biofilm reactor, AcSBBR) was operated with designed synthetic wastewater [organic loading rate (OLR) 4.75 kg COD/m³-day] under acidophilic conditions (pH 6.0) using selectively enriched acidogenic mixed consortia. The resultant outlet from AcSBBR composed of fermentative soluble intermediates (with residual carbon source), was used as feed for subsequent methanogenic bioreactor (methanogenic/anaerobic sequencing batch biofilm reactor, AnSBBR, pH 7.0) to generate additional biogas (CH₄) utilizing residual organic composition employing anaerobic mixed consortia. During the stabilized phase of operation (after 60 days) AcSBBR showed H₂ production of 16.91 mmol/day in association with COD removal efficiency of 36.56% (SDR_A—1.736 kg COD/m³-day). AnSBBR showed additional COD removal efficiency of 54.44% (SDR_M—1.071 kg COD/m³-day) along with CH₄ generation. Integration of the acidogenic and methanogenic processes enhanced substrate degradation efficiency (SDR_T—4.01 kg COD/m³-day) along with generation of both H₂ and CH₄ indicating sustainability of the process.     

A rapid and simple protocol for evaluating biohydrogen production potential (BHP) of wastewater with simultaneous process optimization

Biohydrogen production utilizing negative valued waste through dark-fermentation process is one of the emerging areas. Reported conditions for H₂ production are significantly variable and comparative analysis of data is major problem for unified understanding. A simple, rapid and generalized two phase methodology/protocol was developed to evaluate the biohydrogen production potential (BHP) of negative valued wastewater as substrate/feed-stock for renewable biohydrogen production using mixed consortia. Critical factors that can influence the overall process viz., redox condition, organic load and biocatalyst were considered in the designing the methodology. Feasibility of protocol was initially evaluated with synthetic wastewater and further validated with real field composite food and slaughter house wastewaters. The selected operational factors showed marked influence on both H₂ production and wastewater treatment. The reported methodology/protocol not only provides the ability of selected wastewater to generate H₂ but also facilitates process understanding based on selected factors and finally acquiesce optimum conditions.