Improving net energy gain in fermentative biohydrogen production from glucose

Our previous studies had demonstrated enhanced fermentative hydrogen production from sucrose in batch reactors with dairy manure as a supplement providing nutrients, buffering, and hydrogen-producing organisms. In this study, manure leachate is evaluated as a supplement in glucose fermentation in batch and continuous flow reactors at 25 °C without any nutrient supplements, initial pH adjustments, buffering, or stirring. Hydrogen yields found in this study are comparable to or better than those reported at higher temperatures. When the heat energy expended to maintain the test temperatures is considered, positive net energy gain of ∼10 kJ/L of reactor volume was achieved while most literature reports translated to negative net energy gain. Anaerobic digestion (AD) and microbial fuel cells (MFC) were evaluated as follow-up processes to extract additional energy from the end products of dark fermentation (DF). This evaluation showed that DF followed by MFCs to produce electricity to be a more energy-efficient approach.     

Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation

The effect of different food to microorganism ratios (F/M) (1–10) on the hydrogen production from the anaerobic batch fermentation of mixed food waste was studied at two temperatures, 35 ± 2 °C and 50 ± 2 °C. Anaerobic sludge taken from anaerobic reactors was used as inoculum. It was found that hydrogen was produced mainly during the first 44 h of fermentation. The F/M between 7 and 10 was found to be appropriate for hydrogen production via thermophilic fermentation with the highest yield of 57 ml-H₂/g VS at an F/M of 7. Under mesophilic conditions, hydrogen was produced at a lower level and in a narrower range of F/Ms, with the highest yield of 39 ml-H₂/g VS at the F/M of 6. A modified Gompertz equation adequately (R² > 0.946) described the cumulative hydrogen production yields. This study provides a novel strategy for controlling the conditions for production of hydrogen from food waste via anaerobic fermentation.     

Effect of temperature on anaerobic fermentative hydrogen gas production from feedlot cattle manure using mixed microflora

Hydrogen production from the anaerobic fermentation of feedlot cattle manure was examined in batch cultures over a temperature range from 36 to 60 °C at a pH of 5.2. The amount of hydrogen produced increased with temperature to a maximum of 65 L H₂ kg TS⁻¹ at 52 °C. At temperatures > 52 °C, acetate was the main volatile fatty acid (VFA) accumulated, while at <52 °C butyrate accumulated the most. Formate was detected in the 56 and 60 °C treatments but was absent in all others. Thermophilic conditions resulted in the highest hydrogen production rates, with maximum hydrogen production occurring 52 °C. Changing incubation temperature by small (4 °C) increments up or down from 52 °C resulted in changes in the metabolic flux (conversion of substrate to VFA and gaseous products) of the anaerobic digestion system. These findings indicate that the hydrogen production potential of anaerobic systems utilizing heat treated cattle manure as inoculum is affected greatly by incubation temperature.     

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH₄⁺, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H₂ photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L⁻¹), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H₂ production. The influence of various fermentation end products at different concentrations (up to 30 g L⁻¹) on dark, H₂ production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L⁻¹) resulted in 5.7 L H₂ L⁻¹ of culture (1.6 mol H₂ per mole of hexose) with the co-production mainly of butyrate and acetate.     

Hydrogen gas production from acid hydrolyzed wheat starch by combined dark and photo-fermentation with periodic feeding

Hydrogen gas production from acid hydrolyzed waste wheat starch by combined dark and photo-fermentation was investigated in continuous mode with periodic feeding and effluent removal. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides (NRRL-B 1727) were used as the seed culture for dark and light fermentations, respectively with biomass ratio of Rhodobacter/sludge = 3. Hydraulic residence time (HRT) was changed between 1 and 8 days by adjusting the feeding periods. Ground waste wheat was acid hydrolyzed at pH = 3 and 121 °C for 30 min using an autoclave and the resulting sugar solution was used as the substrate for combined fermentation after pH adjustment and nutrient addition. The highest daily hydrogen gas production (41 ml d⁻¹), hydrogen yield (470 ml g⁻¹ total sugar = 3.4 mol H₂ mol⁻¹glucose), volumetric and specific hydrogen production rates were obtained at the HRT of 8 days. The highest biomass and the lowest total volatile fatty acids (TVFA) concentrations were also realized at HRT = 8 days indicating VFA fermentation by Rhodobacter sp. at high HRTs. The lowest total sugar loading rate of 0.625 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate. Hydrogen gas production by combined fermentation with periodic feeding was proven to be an effective method resulting in high hydrogen yields at long HRTs.     

Dark and acidic conditions for fermentative hydrogen production

Bacterial consortium capable of producing hydrogen in low pH (LpH) range of 3.3–4.3 is reported in this study. This operational pH is two full units below that of previously reported hydrogen producing organisms. Low pH inocula were derived from a batch biohydrogen reactor inoculated with heat treated compost (∼120 °C, 2 h), which was allowed to accumulate biogas to reach three atmospheres of equivalent headspace pressure and system pH of 3.0. Acclimation effect had positive influence on H₂ production and LpH inocula were passed sequentially into more than 15 generations to achieve consistent conversion efficiency and hydrogen composition, further tested in 23 other culture cycles. With hydrogen composition in the headspace ranging from 50% to 60%, conversion efficiency of ∼43% achieved in LpH systems is comparable to that of other buffered systems. Feasibility of hydrogen production in LpH systems is demonstrated in unbuffered reactors under intermittent pressure release conditions and in absence of initial pH adjustment and stirring. Conversion efficiencies, however, decreased by ∼1-fold for each 3 °C drop below the optimum temperature of 22 °C.     

Energy balance of dark anaerobic fermentation as a tool for sustainability analysis

A process aimed at producing energy needs to produce more energy than the energy necessary to run the process itself in order to be energetically sustainable. In this paper, an energy balance of a batch anaerobic bioreactor has been defined and calculated, both for different operative conditions and for different reactor scales, in order to analyze the sustainability of hydrogen production through dark anaerobic fermentation. Energy production in the form of hydrogen and methane, energy to warm up the fermentation broth, energy loss during fermentation and energy for mixing and pumping have been considered in the energy balance. Experimental data and literature data for mesophilic microorganism consortia have been used to calculate the energy balance. The energy production of a mesophilic microorganism consortium in a batch reactor has been studied in the 16–50 °C temperature range. The hydrogen batch dark fermentation resulted to only have a positive net production of energy over a minimal reactor dimension in summer conditions with an energy recovery strategy. The best working temperature resulted to be 20 °C with 20% of available energy. Hydrogen batch dark fermentation may be coupled with other processes to obtain a positive net energy by recovering energy from the end products of hydrogen dark fermentation. As an example, methane fermentation has been considered to energetically valorize the end products of hydrogen fermentation. The combined process resulted in a positive net energy over the whole range of tested reactor dimension with 45–90% of available energy.     

Effect of operation parameters on anaerobic fermentation using cow dung as a source of microorganisms

Anaerobic fermentation by microorganisms is a promising method of hydrogen production for it can be conducted at mild conditions. In this paper, a series of tests were carried out to investigate the effect of pH, hydraulic retention time (HRT), temperature (T) and substrate concentration on anaerobic dark fermentation. Glucose was utilized as model substrate. The Taguchi orthogonal array was applied in the experimental design and a verification experiment was tested. The results showed the optimal parameters for hydrogen production were pH 5.0, HRT 8.34 h, T 33.5 °C and substrate concentration of 14 g/L, with hydrogen yield of 2.15 mol H₂/mol glucose. Butyric-type fermentation occurred in most tests. According to the analysis of effluent contents, at pH 5.5, 5.0, 4.0, the effluent contained mostly butyric acid (43.1–56.6%), followed by acetic acid (24.6–29.8%); at HRT 4.17, 6.26, 8.34 h, the effluent contained mostly butyric acid (43.0–53.6%). Increasing temperature from 29 to 39.5 °C resulted in the decrease of butyric acid percentage but increase of ethanol percentage. Substrate concentration had little effect on product constitution.     

Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone

Ceramic ring and pumice stone were used as a support matrix for the enhancement of biohydrogen production in immobilized cell culture systems. The reactors were continuously operated for the hydrogen fermentation using sucrose as the major carbon source at varying hydraulic retention times (HRT) as an important operational factor. In terms of volumetric hydrogen production, the best value was obtained with ceramic ring at 1.5 h HRT (2.98 l H₂/l/d), on the other hand, the pumice stone packed reactor resulted in 30% less volumetric hydrogen production (2.28 l H₂/l/d) at two fold longer retention time (HRT 3 h). It was demonstrated that volumetric hydrogen production with the immobilized bioreactor configurations was 6 fold better than the suspended culture bioreactor configuration (CSTR). Furthermore, up to 4 mol and 5 mol hydrogen yields per mole of sucrose used (which are 62.5% and 50% of the theoretical values) were achieved by pumice stone and ceramic ring packed reactors, respectively, whereas suspended culture system yielded only 0.5 mol H₂/mol sucrose.     

Hydrogen production by combined dark and light fermentation of ground wheat solution

Ground waste wheat was subjected to combined dark and light batch fermentation for hydrogen production. The dark to light biomass ratio (D/L) was changed between 1/2 and 1/10 in order to determine the optimum D/L ratio yielding the highest hydrogen formation rate and the yield. Hydrogen production by only dark and light fermentation bacteria was also realized along with the combined fermentations. The highest cumulative hydrogen formation (CHF = 76 ml), hydrogen yield (176 ml H₂ g⁻¹ starch) and formation rate (12.2 ml H₂ g⁻¹ biomass h⁻¹) were obtained with the D/L ratio of 1/7 while the lowest CHF was obtained with the D/L ratio of 1/2. Dark–light combined fermentation with D/L ratio of 1/7 was faster as compared to the dark and light fermentations alone yielding high hydrogen productivity and reduced fermentation time. Dark and light fermentations alone also yielded considerable cumulative hydrogen, but slower than the combined fermentation.     

Biohydrogen production from household solid waste (HSW) at extreme-thermophilic temperature (70 °C) – Influence of pH and acetate concentration

Hydrogen production from household solid waste (HSW) was performed via dark fermentation by using an extreme-thermophilic mixed culture, and the effect of pH and acetate on the biohydrogen production was investigated. The highest hydrogen production yield was 257 ± 25 mL/gVS_added at the optimum pH of 7.0. Acetate was proved to be inhibiting the dark fermentation process at neutral pH, which indicates that the inhibition was caused by total acetate concentration not by undissociated acetate. Initial inhibition was detected at acetate concentration of 50 mM, while the hydrogen fermentation was seriously inhibited at acetate concentration of 200 mM. At 200 mM acetate concentration, the hydrogen yield was 36 ± 25 mL/gVS_added, which was almost 7 times lower than the yield of 254 ± 13 mL/gVS_added, which was achieved at lower acetate concentration (5–25 mM). Additional to the negative effect on the hydrogen yield, acetate was resulting in the longer lag phase during batch fermentations. The lag phase was more than 100 h at acetate concentration of more than 150 mM, while it was only 3–4 h at 5–25 mM acetate.     

Production of hydrogen from sewage biosolids in a continuously fed bioreactor: Effect of hydraulic retention time and sparging

Hydrogen was produced from primary sewage biosolids via mesophilic anaerobic fermentation in a continuously fed bioreactor. Prior to fermentation the sewage biosolids were heated to 70 °C for 1 h to inactivate methanogens and during fermentation a cellulose degrading enzyme was added to improve substrate availability. Hydraulic retention times (HRT) of 18, 24, 36 and 48 h were evaluated for the duration of hydrogen production. Without sparging a hydraulic retention time of 24 h resulted in the longest period of hydrogen production (3 days), during which a hydrogen yield of 21.9 L H₂ kg⁻¹ VS added to the bioreactor was achieved. Methods of preventing the decline of hydrogen production during continuous fermentation were evaluated. Of the techniques evaluated using nitrogen gas to sparge the bioreactor contents proved to be more effective than flushing just the headspace of the bioreactor. Sparging at 0.06 L L min⁻¹ successfully prevented a decline in hydrogen production and resulted in a yield of 27.0  L H₂ kg⁻¹ VS added, over a period of greater than 12 days or 12 HRT. The use of sparging also delayed the build up of acetic acid in the bioreactor, suggesting that it serves to inhibit homoacetogenesis and thus maintain hydrogen production.     

Hydrogen storage in Co-and Zn-based metal-organic frameworks at ambient temperature

Hydrogen adsorption properties of some Co-and Zn-based Metal-Organic Framework (MOF) materials were studied at near ambient temperatures. Maximal hydrogen storage capacity of 0.75 wt% was found for a Zn-based material at 175 Bar hydrogen pressure and T = −4 °C. Hydrogen adsorption correlated linearly with BET surface area and strongly depends on temperature. Relatively low structural stability of some MOF's results in framework collapse during degassing and hydrogen adsorption measurements.     

Study of hydrogen physisorption on nanoporous carbon materials of different origin

Hydrogen adsorption capabilities of different nanoporous carbon, i.e. amorphous carbons obtained by chemical activation (with KOH) of a sucrose-derived char previously ground by ball milling and carbon replicas of NH₄-Y and mesocellular silica foam (MSU-F) inorganic templates, were measured and correlated to their porous properties. The porous texture of the prepared carbon materials was studied by means of N₂ and CO₂ adsorption isotherms measured at −196 °C and 0 °C, respectively. Comparison with nanoporous carbons obtained without pre-grinding the sucrose-derived char [12] shows that the ball milling procedure favours the formation of highly microporous carbon materials even at low KOH loadings, having a beneficial effect of the interaction between the char particles and the activating agent. Hydrogen adsorption isotherms at −196 °C were measured in the 0.0-1.1 MPa pressure range, and a maximum hydrogen adsorption capacity of 3.4 wt.% was obtained for the amorphous carbon prepared by activation at 900 °C with a KOH/char weight ratio of 2. Finally, a linear dependence was found between the maximum hydrogen uptake at 1.1 MPa and the samples microporous volume, confirming previous results obtained at −196 °C and sub-atmospheric pressure [12].     

High yield simultaneous hydrogen and ethanol production under extreme-thermophilic (70 °C) mixed culture environment

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO₄ (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H₂/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Biohydrogen production from starch wastewater and application in fuel cell

Biohydrogen was produced from starch in wastewater by anaerobic fermentation. The effects of parameters, such as pH, starch concentration were investigated and optimum operating conditions were determined. The optimal pH and starch concentration for hydrogen production at 37 °C were 6.5 and 5 g/L, respectively with a maximum hydrogen yield of 186 ml/g-starch. The produced biogas contains 99% of hydrogen after passing through KOH solution to remove CO₂. The anaerobic fermentation installation was integrated with a proton-exchange-membrane fuel cell (PEMFC) system for on-line electricity generation. This combination system of biohydrogen and fuel cell achieved a power output of 0.428 W at 0.65 V per cell.     

Hydrogen production for PEM fuel cell by gas phase reforming of glycerol as byproduct of bio-diesel. The use of a Pd-Ag membrane reactor at middle reaction temperature

Glycerol as a byproduct of biodiesel production represents a renewable energy source. In particular, glycerol can be used in the field of hydrogen production via gas phase reforming for proton exchange membrane fuel cell (PEMFC) applications. In this work, glycerol steam reforming (GSR) reaction was investigated using a dense palladium-silver membrane reactor (MR) in order to produce pure (or at least CO-free) hydrogen, using 0.5 wt% Ru/Al₂O₃ as reforming catalyst. The experiments are performed at 400 °C, water to glycerol molar feed ratio 6:1, reaction pressure ranging from 1 to 5 bar and weight hourly space velocity (WHSV) from 0.1 to 1.0 h⁻¹. Moreover, a comparative study is given between the Pd-Ag MR and a traditional reactor (TR) working at the same MR operating conditions. The effect of the WHSV and reaction pressure on the performances of both the reactors in terms of glycerol conversion and hydrogen yield is also analyzed. The MR exhibits higher conversion than the TR (∼60% as best value for the MR against ∼40% for the TR, at WHSV = 0.1 h⁻¹ and 5 bar), and high CO-free hydrogen recovery (around 60% at WHSV = 0.1 h⁻¹ and 5 bar). During reaction, carbon coke is formed limiting the performances of the reactors and inhibiting, in particular, the hydrogen permeation through the membrane with a consequent reduction of hydrogen recovery in the permeate side.     

Thermodynamics of hydrogen production by the steam reforming of butanol: Analysis of inorganic gases and light hydrocarbons

The thermodynamics of butanol steam reformation for the production of hydrogen were simulated using a Gibbs free-energy-minimisation method with water–butanol molar feed ratios (WBFR) between 1 and 18, a pressure range of 1–50 bar and reaction temperatures from 300 to 900 °C. The differences in H₂ and CO production were calculated as functions of WBFR and temperature at 1 bar. On the basis of the equilibrium calculations with higher-hydrocarbon compounds excluded, the optimal operating conditions obtained were 600–800 °C, 1 bar and WBFR = 9–12. At these conditions, the yield of hydrogen and carbon monoxide was maximised and methane selectivity minimised. The yield of hydrogen was in the range of 75.13–81.27% (wet basis) with selectivities of 46.20–54.96%. This was achieved at a temperature of 800 °C and WBFR from 9 to 12. Carbon monoxide yield ranged between 65.48 and 55.57% (wet basis), with selectivities ranging from 14.56 to 10.66%. The formation of coke was completely inhibited at these operating conditions. In order to evaluate the effect of methane on coke formation at lower temperatures, simulations were performed in two sets, i.e., primary products (H₂, CO, CO₂ and C) including or excluding methane. The results indicate that some coke can be hydrogenated to methane at 300 °C and WBFR = 3, and that higher pressure favours hydrogenation reactions. Higher pressure had a negative effect on hydrogen and carbon monoxide yields.