Influence of C/P and C/N ratios and microbial characterization in hydrogen and ethanol production in an anaerobic fluidized bed reactor

The aim of this study was to evaluate the influence of C/P and C/N ratios on the production of hydrogen and ethanol in four anaerobic fluidized bed reactors: R1 (C/N = 100), R2 (C/N = 150), R3 (C/N = 200), and R4 (C/N = 250). The hydraulic retention time (HRT) was maintained at 2 h, and the C/P ratios varied from 300 to 1100. Reactors were filled with grounded tire and fed with synthetic substrate containing glucose (5000 mg L^?1). The effluent pH was around 3.7. The highest values for hydrogen yield (HY) and hydrogen production rate (HPR) were obtained at a C/P = 700 ratio in all reactors. The best performance was achieved at R3 (C/N = 200): HY of 0.76 mol H₂ mol^?1 glucose and HPR of 0.70 L h^?1 L^?1. The highest value for ethanol yield was obtained at C/P = 700 in R1 (1.5 mol EtOH mol^?1 glucose). Ethanol- and hydrogen-producing fermenters, such as Ethanoligenens sp. and Clostridium sp. were identified by molecular analysis. Lactobacillus sp. was also identified in this study.     

Hydrogen and methane production potential of agave bagasse enzymatic hydrolysates and comparative technoeconomic feasibility implications

In the present work, agave bagasse enzymatic hydrolysates obtained with newly locally-available commercial enzymatic preparations were explored for their corresponding hydrogen and methane production potential in batch mode. The major levels in chemical oxygen demand and total carbohydrates were provided by enzymatic hydrolysates made with Zymapect and Stonezyme, respectively. Batch experiments demonstrated that Celluclast 1.5L achieves the maximum hydrogen productivity (1.88 L H₂/L), from 1.6 to 2.0-fold higher than other alternatives, whereas Zymapect attains the highest methane productivity (1.32 L CH₄/L), with high specific yield reached by both Stonezyme and Zymapect (162 and 163 L CH₄/kg bagasse), from 1.7 to 2.0-fold higher than other options. Finally, a preliminary techno-economic analysis allowed to elucidate that the cheapest alternatives for hydrogen and methane production at batch scale are Celluclast 1.5L and Stonezyme, respectively. Overall, the present analysis could serve as groundwork for the selection of the best enzymatic alternatives for hydrogen and methane production.     

Hydrogen production from acid and enzymatic oat straw hydrolysates in an anaerobic sequencing batch reactor: Performance and microbial population analysis

Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H₂/mol sugar consumed) and hydrogen production rate (HPR, 278 mL H₂/L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H₂/mol sugar consumed and 29.6 mL H₂/L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.     

Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae

The newspaper is comprised of (w w^?1) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various enzymatic treatments using cellulase, xylanase and laccase, cellulase enzyme system was found to deink the newspaper most efficiently. The saccharification of deinked paper pulp using enzyme cocktail containing exoglucanase (20 U g^?1), β-glucosidase (60 U g^?1) and xylanase (80 U g^?1) resulted in 59.8% saccharification. Among additives, 1% (v v^?1) Tween 80 and 10 mol m^?3 CoCl₂ improved the enzymatic hydrolysis of newspaper maximally, releasing 14.64 g L^?1 sugars. The fed batch enzymatic saccharification of the newspaper increased the sugar concentration in hydrolysate from 14.64 g L^?1 to 38.21 g L^?1. Moreover, the batch and fed batch enzymatic hydrolysates when fermented with Saccharomyces cerevisiae produced 5.64 g L^?1 and 14.77 g L^?1 ethanol, respectively.     

Biodegradation of Potamogeton pectinatus biomass by hydrogen and methane coproduction: Effect of different pretreatments and parallel factor analysis

To enhance the hydrogen and methane coproduction potential, three pretreatments (i.e., acid, alkali and cellulase) were investigated during a two-stage anaerobic fermentation using Potamogeton pectinatus biomass. The fluorescence spectral characteristics of the dissolved organic matter (DOM) from the two-stage effluents, coupled with parallel factor analysis, were studied. The maximum hydrogen proportions (42.65%) and production rate (4.1 mL h^?1) were obtained under the 0.5 mol L^?1 HCl pretreatment. The highest methane proportions (52.82%) and production rate (14.2 mL h^?1) were observed under the 0.5 mol L^?1 HCl and 10 mg g^?1 cellulase pretreatments, respectively. Combined with fluorescence spectra and parallel factor analysis, three fluorescent components were identified, and the protein-like substances were determined to be dominant. Using the acid pretreatment, the change of the maximum fluorescence intensities in the DOM was the most significant among the three pretreatments, followed by that of the cellulose pretreatment. The result indicated that the macromolecular substances in P. pectinatus can be decomposed by effective pretreatment and thereby enhances the hydrogen and methane coproduction potential. This technique represents a promising method for improving cellulosic biomass biodegradation and green energy coproduction.     

Biological hydrogen production from sugar beet molasses by agar immobilized R. capsulatus in a panel photobioreactor

Biohydrogen production from sugar beet molasses was investigated by using agar immobilized R. capsulatus YO3. A panel photobioreactor (1.4 L) was employed for a long-term hydrogen production in both indoor and outdoor conditions. The impact of several initial molasses concentrations on hydrogen production, yield and productivity were assessed. Indoor studies revealed that initial sucrose concentration in molasses should be kept below 20 mM to prevent inhibition of hydrogen production. The highest hydrogen productivity of 0.64 ± 0.06 mmol H₂ L^?1 h^?1 and yield of 12.2 ± 1.5 mol H₂/mol sucrose were obtained in indoors throughout 20 days of operation. For outdoors, hydrogen production continued for 40 days including consecutive 10 rounds under natural outdoor conditions. In outdoor conditions, the maximum hydrogen productivity and yield were 0.79 ± 0.04 mmol H₂ L^?1 h^?1 and 5.2 ± 0.4 mol H₂/mol sucrose respectively. These results indicate that the proposed system is promising for biohydrogen production from molasses at large-scale natural conditions.     

Enhancing saccharification of Agave tequilana bagasse by oxidative delignification and enzymatic synergism for the production of hydrogen and methane

Agave tequilana bagasse is a suitable lignocellulosic residue for energy production. However, the presence of lignin and the heterogeneous structure of hemicellulose may hinder the availability of polysaccharides. In this work, the pretreatment of A. tequilana bagasse with alkaline hydrogen peroxide (AHP) followed by enzymatic saccharification with hemicellulases and cellulases was assessed for the removal of lignin and extraction of fermentable sugars, respectively. Results of the AHP pretreatment indicated that it is possible to attain up to 97% delignification and recover 88% of cellulose and hemicellulose after only 1.5 h of treatment. Regarding the saccharification process, the total sugar yield and productivity were both increased by 2-fold using an enzymatic mixture (cellulases + hemicellulases) compared to single enzyme hydrolysis (cellulases), evidencing synergism. Further evaluation of the hydrolyzates as substrate for hydrogen and methane production, resulted in yields 1.5 and 3.6-times (215.14 ± 13 L H₂ and 393.4 ± 13 L CH₄ per kg bagasse, respectively) superior to those obtained with hydrolyzates of non-pretreated bagasse processed with a single enzyme. Overall, using AHP pretreatment and subsequent hydrolysis with enzymatic mixtures improves the saccharification of A. tequilana bagasse enhancing the production of hydrogen and methane.     

Scale-up studies for stable,long-term indoor and outdoor production of hydrogen by immobilized Rhodobacter capsulatus

Photofermentative hydrogen production by immobilized Rhodobacter capsulatus YO3 was carried out in a novel photobioreactor in sequential batch mode under indoor and outdoor conditions. Long-term H₂ production was realized in a 1.4 L photobioreactor for 64 days using Rhodobacter capsulatus YO3 immobilized with 4% (w/v) agar on 5 mM sucrose and 4 mM glutamate. The highest hydrogen yield (19 mol H₂/mol sucrose) and hydrogen productivity (0.73 mmol H₂ L^?1 h^?1) were achieved indoors on 5 mM sucrose. The effect of initial sucrose concentration (5 mM, 10 mM, and 20 mM) on hydrogen production was also investigated. Sustained hydrogen production was carried out under natural, outdoor conditions as well. For the outdoor experiments, the highest hydrogen productivity and yield were obtained as 0.87 ± 0.06 mmol H₂ L^?1 h^?1 and 6.1 ± 0.2 mol H₂/mol sucrose, respectively on 10 mM sucrose. Furthermore, this system prevented sudden pH drops and fluctuations caused by the utilization of sucrose throughout the process. These results demonstrate that a proper immobilization setup can lead to long-term efficient and robust hydrogen production even under naturally varying conditions.     

Assessment of the adequacy of different Mediterranean waste biomass types for fermentative hydrogen production and the particular advantage of carob (Ceratonia siliqua L.) pulp

The conversion of agro-industrial byproducts, residues and microalgae, which are representative or adapted to the Mediterranean climate, to hydrogen (H₂) by C. butyricum was compared. Five biomass types were selected: brewery’s spent grain (BSG), corn cobs (CC), carob pulp (CP), Spirogyra sp. (SP) and wheat straw (WS). The biomasses were delignified and/or saccharified, except for CP which was simply submitted to aqueous extraction, to obtain fermentable solutions with 56.2–168.4 g total sugars L^?1. In small-scale comparative assays, the H₂ production from SP, WS, CC, BSG and CP reached 37.3, 82.6, 126.5, 175.7 and 215.8 mL (g biomass)^?1, respectively. The best fermentable substrate (CP) was tested in a pH-controlled batch fermentation. The H₂ production rate was 204 mL (L h)^?1 and a cumulative value of 3.9 L H₂ L^?1 was achieved, corresponding to a H₂ production yield of 70.0 mL (g biomass)^?1 or 1.6 mol (mol of glucose equivalents)^?1_. The experimental data were used to foresight a potential energy generation of 2.4 GWh per year in Portugal, from the use of CP as substrate for H₂ production.     

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h^?1 and the average pseudo-steady state hydrogen production rate was 36 mL H₂ L^?1 medium h^?1. The cumulative hydrogen production was 430 mL H₂ L^?1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H₂ L^?1 h^?1 and the cumulative hydrogen production was 517 mL H₂ L^?1 medium. Reducing the feed rate to less than 48 mL h^?1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.     

Optimization of dilute acid and enzymatic hydrolysis for dark fermentative hydrogen production from the empty fruit bunch of oil palm

Pretreatment of the empty fruit brunch (EFB) from oil palm was investigated for H₂ fermentation. The EFB was hydrolyzed at various temperatures, H₂SO₄ concentrations, and reaction times. Subsequently, the acid-hydrolysate underwent enzymatic saccharification under various temperature, pH, and enzymatic loading conditions. Response surface methodology derived the optimum sugar concentration (SC), hydrogen production rate (HPR), and hydrogen yield (HY) as 28.30 g L⁻¹, 2601.24 mL H₂ L⁻¹d⁻¹, and 275.75 mL H₂ g⁻¹ total sugar (TS), respectively, at 120 °C, 60 min of reaction, and 6 vol% H₂SO₄, with the combined severity factor of 1.75. Enzymatic hydrolysis enhanced the SC, HY, and HPR to 34.52 g L⁻¹, 283.91 mL H₂ g⁻¹ TS, and 3266.86 mL H₂ L⁻¹d⁻¹, respectively, at 45 °C, pH 5.0, and 1.17 mg enzyme mL⁻¹. Dilute acid hydrolysis would be a viable pretreatment for biohydrogen production from EFB. Subsequent enzymatic hydrolysis can be performed if enhanced HPR is required.     

A new side-looking at the dark fermentation of sugarcane vinasse: Improving the carboxylates production in mesophilic EGSB by selection of the hydraulic retention time and substrate concentration

In recent years, a lot of scientific effort has been put into reusing the energy potential of sugarcane vinasse by dark fermentation. However, the findings so far indicate that new pathways need to be followed. In this context, this study assessed the effect of hydraulic retention time (HRT, from 24 to 1 h) on vinasse fermentation (10, 20, and 30 g COD L^?1) in three mesophilic expanded granular sludge bed reactors (EGSB). The carbohydrate conversion remained above 60% at all organic loading rates applied. The maximum hydrogen production rate (8.77 L day^?1 L^?1) was obtained for 720 kg COD m^?3 day^?1 and associated to the lactate-acetate pathway. The highest productivities of propionic, acetic, and butyric acids were 3.11, 1.68, and 2.45 g L^?1 h^?1, respectively, at a HRT of 1 h. At this HRT, the degrees of acidification remained between 54% and 76% in all EGSB reactors. This research provides insights for carboxylate production from sugarcane vinasse and suggests applying the EGSB setup in the acidogenic stage of two-stage processes.     

Hydrogen production by steam methane reforming in a membrane reactor equipped with a Pd composite membrane deposited on a porous stainless steel

With the aim of producing hydrogen at low cost and with a high conversion efficiency, steam methane reforming (SMR) was carried out under moderate operating conditions in a Pd-based composite membrane reactor packed with a commercial Ru/Al₂O₃ catalyst. A Pd-based composite membrane with a thickness of 4–5 μm was prepared on a tubular stainless steel support (diameter of 12.7 mm, length of 450 mm) using electroless plating (ELP). The Pd-based composite membrane had a hydrogen permeance of 2.4 × 10^?3 mol m^?1 s^?1 Pa^?0.5 and an H₂/N₂ selectivity of 618 at a temperature of 823 K and a pressure difference of 10.1 kPa. The SMR test was conducted at 823 K with a steam-to-carbon ratio of 3.0 and gas hourly space velocity of 1000 h^?1; increasing the pressure difference resulted in enhanced methane conversion, which reached 82% at a pressure difference of 912 kPa. To propose a guideline for membrane design, a process simulation was conducted for conversion enhancement as a function of pressure difference using Aspen HYSYS^®. A stability test for SMR was conducted for ～120 h; the methane conversion, hydrogen production rate, and gas composition were monitored. During the SMR test, the carbon monoxide concentration in the total reformed stream was <1%, indicating that a series of water gas shift reactors was not needed in our membrane reactor system.     

Continuous hydrogen production from cofermentation of sugarcane vinasse and cheese whey in a thermophilic anaerobic fluidized bed reactor

The co-fermentation of vinasse and cheese whey (CW) was evaluated in this study by using two thermophilic (55° C) anaerobic fluidized bed reactors (AFBRs). In AFBR using vinasse and CW (AFBR-V-CW), the CW was added in increasing proportions (2, 4, 6, 8, and 10 g COD.L^?1) to vinasse (10 g COD.L^?1) to assess the advantage of adding CW to vinasse. By decreasing the hydraulic retention time (HRT) from 8 h to 1 h in AFBR-V, maximum hydrogen yield (HY), production rate (HPR), and H₂ content (H₂%) of 1.01 ± 0.06 mmol H₂.g COD^?1, 2.54 ± 0.39 L H₂.d^?1.L^?1, and 47.3 ± 2.9%, respectively, were observed at an HRT of 6 h. The increase in CW concentration to values over 2 g COD.L^?1 in AFBR-V-CW decreased the HY, PVH, and H₂%, with observed maximum values of 0.82 ± 0.07 mmol H₂.g COD^?1, 1.41 ± 0.24 L H₂.d^?1.L^?1, and 55.5 ± 3.7%, respectively, at an HRT of 8 h. The comparison of AFBR-V-CW and AFBR-V showed that the co-fermentation of vinasse with 2 g COD.L^?1 of CW increased the HPR, H₂%, and HY by 117%, 68%, and 82%, respectively.     

Selection of metabolic pathways for continuous hydrogen production under thermophilic and mesophilic temperature conditions in anaerobic fluidized bed reactors

This study evaluated the influence of hydraulic retention time (HRT) on hydrogen (H₂) production in anaerobic fluidized bed reactors at mesophilic (30 °C, AFBR-M) and thermophilic (55 °C, AFBR-T) temperatures. Reactors were fed sucrose-based synthetic wastewater (5000 mg chemical oxygen demand·L^?1) in the HRT of 8, 6, 4, 2, or 1 h. H₂ production rate increased from 67.8 ± 14.8 to 194.9 ± 57.0 ml H₂·h^?1 L^?1 (AFBR-T) and from 72.0 ± 10.0 to 344.4 ± 74.0 mL H₂·h^?1·l^?1 (AFBR-M) when HRT decreased from 8 to 1 h. Maximum H₂ yields for AFBR-T and AFBR-M were 1.93 ± 0.21 and 2.68 ± 0.48 mol H₂·mol^?1 sucrose, respectively. The main metabolites were acetic acid (31.3%–41.5%) and butyric acid (10.2%–20.7%) (AFBR-M) and acetate (20.1%–39.3%) and ethanol (14.3%–29.9%) (AFBR-T). Denaturing gradient gel electrophoresis profiles revealed selective enrichment of microbial populations responsible for H₂ production by the aceto-butyric route (AFBR-M) and ethanol-type fermentation (AFBR-T).     

Enhancement of fermentative hydrogen production by Thermotoga maritima through hyperthermophilic anaerobic co-digestion of fruit-vegetable and fish wastes

In this work, different proportions of model fruit and vegetable wastes (MFVW) and acid hydrolyzed fish wastes (AHFW) were used for hydrogen production in a minimum culture medium based on seawater. Experiments were performed in pH-controlled Stirred Tank Reactor (STR) with or without the addition of nitrogen and sulfur sources. The total H₂ production and the maximum hydrogen productivity of T. maritima in the culture medium, containing MFVW and AHFW (45 mmol L^?1 carbohydrates) at a C/N ratio of 12, were 132 mmol L^?1 and 15 mmol h^?1 L^?1, respectively. However, tripling the concentration of carbohydrates to reach a C/N ratio of 22, has increased two times the maximum H₂ productivity (28 mmol h^?1 L^?1) due to the improvement in nutrient balance. The cumulative H₂ production was 285 mmol L^?1, yielding a potential energy generation of 0.1210³ MJ ton^?1 wastes, which could be an interesting alternative for energy recovery.     

Economic evaluation with sensitivity and profitability analysis for hydrogen production from water electrolysis in Korea

Economic evaluation for water electrolysis compared to steam methane reforming has been carried out in terms of unit hydrogen production cost analysis, sensitivity analysis, and profitability analysis to assess current status of water electrolysis in Korea. For a hydrogen production capacity of 30 Nm³ h^?1, the unit hydrogen production cost was 17.99, 16.54, and 20.18 $ kg H₂^?1 for alkaline water electrolysis (AWE), PEM water electrolysis (PWE), and steam methane reforming (SMR), respectively with 11.24, 10.66, and 11.80 for 100 Nm³ h^?1 and 8.12, 7.72, and 7.59 $ kg H₂^?1 for 300 Nm³ h^?1. With sensitivity analysis (SA), the most influential factors on the unit hydrogen production cost depending on the hydrogen production capacity were determined. Lastly, profitability analysis (PA) presented a discounted payback period (DPBP), net present value (NPV), and present value ratio (PVR) for a different discount rate ranging from 2 to 14% and it was found that a discounted cash flow rate of return (DCFROR) was 14.01% from a cash flow diagram obtained for a hydrogen production capacity of 30 Nm³ h^?1.     

Performance of batch solid state fermentation for hydrogen production using ground wheat residue

Ground wheat (21 g) was subjected to batch solid state dark fermentation for bio-hydrogen production. Clostridium acetobutylicum (B-527) was used as the culture of dark fermentation bacteria at mesophilic conditions. Effects of moisture content on the rate and yield of bio-hydrogen formation were investigated. The highest CHF (1222 ml), hydrogen yield (63 ml H₂ g^?1 starch), formation rate (10.64 ml H₂ g^?1 starch h^?1) and specific hydrogen formation rate (0.28 ml H₂ g^?1 biomass h^?1) were obtained with a moisture content of 80%. Nearly complete starch hydrolysis and glucose fermentation were achieved with more than 80% moisture content and the highest substrate conversion rate (21.9 mg L^?1 h^?1) was obtained with 90% moisture content at batch solid state fermentation producing volatile fatty acids (VFA) and H₂.     

Initial pH influences in-batch hydrogen production from scotta permeate

We studied the influence of initial pH on hydrogen (H₂) production using permeate from scotta (a partially deproteinized cheese whey from ricotta production) as substrate (51 g L^?1 lactose). Dark fermentation was carried out at 35 °C in laboratory batch reactors, in an unbuffered system. Hydrogen production and metabolite (volatile fatty acids, ethanol, and lactic acid) evolution during a 96-h period were monitored in reactors with initial pH varying in the range 4–10. In all reactors, H₂ production started only when pH fell below 6. However, it was much higher (+31%) in the reactors with initial alkaline pH. We conclude that H₂ production occurs only at acidic pH values, but initial alkaline pH values increase the overall H₂ production in dark fermentation of lactose-rich substrates.     

Strategies to improve the biohydrogen production from cassava wastewater in fixed-bed reactors

The biological production of hydrogen from cassava starch wastewater (CSW) was evaluated in an anaerobic fixed-bed reactor (UAFBR). The assays were carried out to evaluate the effects of organic loading rate (OLR) increase and strategies of inoculation (AS – anaerobic sludge thermally treated and NF – naturally fermented cassava starch wastewater) on UAFBR performance. The OLR increase (10–20 g L⁻¹ d⁻¹) associated with hydraulic retention time (HRT) decrease (4–2 h) improved the volumetric hydrogen production rate (VHPR, from 229 to 550 mLH₂.L⁻¹.d⁻¹), molar hydrogen flow rate (MHFR, from 1.0 to 2.5 mmolH₂.h⁻¹) and hydrogen yield (HY, from 0.2 to 0.3 molH₂.mol⁻¹Carb) from CSW due to increase in substrate availability. Both inoculation alternatives (AS and NF) were effective for the selection of acidogenic microorganisms, which demonstrates that NF could be considered a simple and economic alternative for the acquisition of inoculum for continuous acidogenic reactors. Hydrogen production decreased after 10 days of operation when the specific organic loading rate (SOLR) reached reduced values (<1 gCarb.g⁻¹VSS.d⁻¹), which impairs hydrogen production. For all assays, methane was present in the biogas after the 20th day of operation mainly due to biomass accumulation, which alters the biota of the reactor. Although many factors could influence the process performance in UAFBR for the production of biohydrogen, the accumulation of biomass have been pointed as the main factor in the determination of the production time, thus demanding the implementation of systematic practices to remove the excess of biomass to maintain the SOLR in levels adequate for hydrogen production.