Hydrogen fermentation of different galactose–glucose compositions during various hydraulic retention times (HRTs)

This study investigated the effects of sugar composition and hydraulic retention time (HRT) on continuous hydrogen fermentation. Continuously-stirred tank reactors (CSTRs) were inoculated with heat-treated digester sludge and fed with 15 g/L of glucose, galactose and galactose: glucose mixture (8:2 ratio-simulating the hydrolysate composition of macroalgae) at HRTs of 6–24 h. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 4.49 L/L/d and 1.62 mol/mol glucose_added were attained while using glucose as feedstock at HRTs of 6 and 18 h, respectively. Meanwhile, galactose provided a peak HPR and HY of 2.35 L/L/d and 1.00 mol/mol galactose_added during the HRTs of 12 and 24 h, respectively. In case of mixed sugars (galactose 8: glucose 2) the production performances were inferior to the sole sugar conditions due to the low substrate utilization of less than 65%, which showed a maximal HPR and HY of 2.75 L/L/d and 0.48 mol/mol carbohydrate_added at the HRTs of 6 and 18 h, respectively.     

Evaluation of process performance on biohydrogen production in continuous fixed bed reactor (C-FBR) using acid algae hydrolysate (AAH) as feedstock

In this study, a novel inoculation method to mitigate the inhibition of 5-hydroxymethylfurfural (5-HMF) is proposed. Acid algae hydrolysate containing 1.5 g 5-HMF/L and 15 g hexose/L hexose was fed to a continuous fixed bed reactor (C-FBR) partially packed with hybrid-immobilized beads. The inoculation method enabled a high rate of H₂ production, due to the reduction of 5-HMF inhibition and enhanced biofilm formation. Maximum hydrogen production was achieved at a hydraulic retention time of 6 h with a hydrogen production rate (HPR) of 20.0 ± 3.3 L H₂/L-d and a hydrogen yield (HY) of 2.3 ± 0.4 mol H₂/mol hexose _added. Butyrate and acetate were the major soluble metabolic products released during fermentation. Quantitative real-time polymerase chain reaction analysis revealed that Clostridium butyricum comprised 94.3% of the total bacteria, which was attributed to the high rate of biohydrogen production.     

Continuous biogenic hydrogen production from dilute acid pretreated algal hydrolysate using hybrid immobilized mixed consortia

This study investigated the bioconversion of dilute acid (2% H₂SO₄) pretreated red algae (Gelidium amansii) hydrolysate into H₂ by anaerobic fermentation in a continuous stirred tank reactor under mesophilic conditions using hybrid immobilized cells as microbial catalyst. Two different hydraulic retention times (HRT) of 24 h and 16 h with a feed concentration of 15 g/L hexose equivalent have been investigated over 85 days of operation to evaluate H₂ production performance and stability of the continuous system. The highest hydrogen production rate (HPR) and hydrogen yield (HY) of 2.7 L/L/d and 1.3 mol/mol substrate hexose_added was achieved at 24 h HRT, while further operation at 16 h HRT led to a significant drop in the hydrogen production with a HPR and HY values of 1.8 L/L/d and 0.7 mol/mol substrate hexose_added, respectively. The bacterial community analysis characterized by 454 pyrosequencing revealed that the changes in HRT significantly influence the composition of the dominant microflora. At longer HRT (24 h), the phyla Firmicutes was abundant over 98%, whereas at shorter HRT (16 h), Proteobacteria being the dominant populations with 84%. These outcomes suggested that controlling appropriate HRT is prerequisite for efficient hydrogen production.     

Dark fermentative hydrogen production from sucrose and molasses

There are many factors affecting the dark fermentative hydrogen production. The interaction of these factors, that is, their combined effects, should be investigated for better design of the systems with stable and higher hydrogen yields. This study aimed to investigate the combined effects of initial substrate, pH, and biomass (or initial substrate to biomass) values on hydrogen production from sucrose and sugar‐beet molasses. Therefore, optimum initial chemical oxygen demand (COD), pH, and volatile suspended solids (VSS) or initial substrate to biomass (VSS) ratio (S/X_o) values leading to the highest dark fermentative hydrogen production were investigated in batch reactors. An experimental design approach (response surface methodology) was used. Results revealed that when sucrose was the substrate, maximum hydrogen production yield (HY) of 2.3 mol H₂/mol sucrose_added was obtained at initial pH of 7 and COD of 10 g/L. Initial S/X_o values studied (4–20 g COD/g VSS) had no effect on HY, while the initial pH was found as the parameter mostly affecting both HY and hydrogen production rate (HPR). When substrate was molasses, initial COD concentration was the only variable affecting HY and HPR. Maximum of both was achieved at 10 g/L initial COD. Initial VSS values studied (2.5–7.5 g/L) had no effect on HPR and HY. This study also indicated that molasses leads to homoacetogenesis for potentially containing intrinsic microorganism and/or natural constituents; thus, sucrose is more advantageous for hydrogen production via fermentation. Homoacetogenesis should be prevented for effective optimization via response surface methodology, if substrate is a natural carbon source potential to have intrinsic microorganisms. Copyright © 2017 John Wiley & Sons, Ltd.     

Simultaneous utilization of galactose and glucose by Saccharomyces cerevisiae mutant strain for ethanol production

Red algal biomass is a promising alternative feedstock for bioethanol production, due to several advantages including high carbohydrate content, growth rate, ethanol yield, and CO₂ fixation ability. However, it has been known that most yeast strains can not utilize galactose, the major sugar of red algae, as efficiently it can utilize glucose. The authors report a novel ethanogenic strain capable of fermenting galactose, Saccharomyces cerevisiae. This mutant yeast strain exhibited exceptional fermentative performance on galactose and a mixture of galactose and glucose. At 120 g/L of initial galactose concentration, ethanol concentration reached 6.9% (v/v) within 36 h with 88.3% of theoretical ethanol yield (0.51 g ethanol/g galactose). The ethanol concentration and yield were higher than that for glucose at the same initial concentration. In a mixed sugar (galactose + glucose) condition, the existence of glucose retarded galactose utilization however, 120 g/L of the mixed sugar was completely consumed within 60 h at any galactose concentration. The critical inhibitory levels of formic acid, levulinic acid and 5-hydroxymethylfurfural (5-HMF) on ethanol fermentation were 0.5, 2.0, and 10.0 g/L; respectively. From this result, the ethanol fermentation efficiency of the novel S. cerevisiae strain using the galactose base of red algae was superior to the fermentation efficiency when using the wild type strain, and the novel strain was found to have resistance to the major inhibitors generated during the saccharification process.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Effects of 5-hydromethylfurfural,levulinic acid and formic acid,pretreatment byproducts of biomass,on fermentative H2 production from glucose and galactose

In this study, the effects of 5-hydromethylfurfural (5-HMF), levulinic acid, and formic acid on fermentative H₂ production have been investigated. The major byproducts of physicochemical pretreatment of lignocellulosic or algal biomass inhibited H₂ production from glucose and galactose. The half maximal inhibitory concentrations (IC₅₀) of 5-HMF on glucose, levulinic acid on glucose, formic acid on glucose, levulinic acid on galactose and formic acid on galactose were 0.59, 1.55, 12.50, 1.33 and 2.99 g/L, respectively. The pretreatment byproducts levels should be mitigated by controlling the severity of a physicochemical treatment or an additional selective removal process prior to H₂ fermentation.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Continuous hydrogen production from tofu processing waste using anaerobic mixed microflora under thermophilic conditions

We carried out continuous fermentative H₂ production from tofu (soybean curd)-processing waste (TPW) using anaerobic mixed microflora under thermophilic (60 °C) conditions and compared the rates and yields of H₂ production in a continuous stirred-tank reactor (CSTR) and a membrane bioreactor (MBR), wherein the membrane filtration unit was coupled to the CSTR. The TPW was diluted with tap water and then hydrolyzed by blending for 5 min in the presence of 0.5% HCl, and it was found that this protocol significantly increased the amount of soluble material in the mixture. The soluble chemical oxygen demand (SCOD)-to-total COD (TCOD) ratio jumped from 14% to 60%, and the soluble carbohydrate concentration was increased threefold, from 2.4 g/L to 7.2 g/L. Accordingly, H₂ production potential was increased 2.8-fold. In a CSTR operation using pretreated TPW as the substrate, a stable volumetric H₂ production rate (VHPR) of 8.17 ± 0.32 L H₂/L/d and a H₂ yield of 1.20 ± 0.05 mol H₂/mol hexose_added at 8-h HRT were achieved. Substantial increases in the VHPR and H₂ yield over those obtained with the CSTR were observed in the MBR operation. The role of the MBR was to increase the retention time of the solid substrate and the concentration of microorganisms, thereby enhancing the substrate utilization rate for H₂ production. Acetic and butyric acids were the main liquid-state metabolites produced during the fermentation process, thus indicating that the thermophilic operation provided favorable conditions for H₂ production from TPW. A maximum H₂ yield of 1.87 mol H₂/mol hexose_added was achieved at 8-h HRT and then gradually decreased to 1.00 mol H₂/mol hexose-equivalent at 2-h HRT. Meanwhile, the VHPR continuously increased to a maximum of 19.86 L H₂/L/d at 4-h HRT and then decreased with a high dilution rate as the HRT was lowered to 2 h (minimum). At 2-h HRT, the degradation of soluble carbohydrate was limited.     

Impact of 5-hydroxy methyl furfural on continuous hydrogen production from galactose and glucose feedstock with periodic recovery

A continuous stirred tank reactor (CSTR) was operated for more than 120 days with fixed hydraulic retention time of 6 h at mesophilic temperature along with a periodic recovery phase towards hydrogen production and stimulated by the existence of 5-hydroxy methyl furfural concentration (5-HMF). Interestingly, CSTR mixed with a small amount of 5-HMF, range of 0.3–0.6 g/L showed at least 50% higher hydrogen production rate than control without 5-HMF. However, when 5-HMF concentration was higher than 0.6 g/L, the performance was significantly inhibited. The bacterial community shifted by 5-HMF from Clostridium-dominated to Lactobacillus-dominated population. Regardless of the remain 5-HMF concentration in CSTR, the microbial community and hydrogen producing performance were restored by stop mixing the 5-HMF from the feedstock. The high-rate hydrogen production of 20.0 ± 1.8 L H₂/L/d was achieved in the presence of 5-HMF using the threshold information and recovery strategy.     

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

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

Optimization of batch dilute-acid hydrolysis for biohydrogen production from red algal biomass

Marine algae are promising alternative sources for bioenergy including hydrogen. Their polymeric structure, however, requires a pretreatment such as dilute-acid hydrolysis prior to fermentation. This study aimed to optimize the control variables of batch dilute-acid hydrolysis for dark hydrogen fermentation of algal biomass. The powder of Gelidium amansii was hydrolyzed at temperatures of 120–180 °C, solid/liquid (S/L) ratios of 5–15% (w/v), and H₂SO₄ concentrations of 0.5–1.5% (w/w), and then fed to batch hydrogen fermentation. Among the three control variables, hydrolysis temperature was the most significant for hydrogen production as well as for hydrolysis efficiency. The maximum hydrogen production performance of 0.51 L H₂/L/hr and 37.0 mL H₂/g dry biomass was found at 161–164 °C hydrolysis temperature, 12.7–14.1% S/L ratio, and 0.50% H₂SO₄. The optimized dilute-acid hydrolysis would enhance the feasibility of the red algal biomass as a suitable substrate for hydrogen fermentation.     

Effect of food to microorganisms (F/M) ratio on biohythane production via single-stage dark fermentation

Hythane is a mixture of hydrogen and methane gases which are generally produced in separate ways. This work studied mesophilic biohythane gas (H₂+CH₄+CO₂) production in a bioreactor via single-stage dark fermentation. The fermentation was conducted in batch mode using mixed anaerobic microflora and food waste and condensed molasses fermentation soluble to elucidate the effects of food to microorganisms (F/M) ratio (ranging from 0.2 to 38.2) on gas production, metabolite variation, kinetics and biohythane-composition indicator performances. The experimental results indicate that the F/M ratio and fermentation time affect biohythane production efficiency with values of peak maximum hydrogen production rate 9.60 L/L-d, maximum methane production rate 0.72 L/L-d, and hydrogen yield (HY) of 6.17 mol H₂/kg COD_added. Depending on the F/M ratios, the H₂, CH₄ and CO₂ biogas components were 10–60%, 5–20% and 35–70%, respectively. Prospects for the further real application for single-stage biohythane fermentation based on the experimental data are proposed. This work characterizes an important reactor operation factor F/M ratio for innovative single-stage dark fermentation.     

Hydrogen production using sono-biohydrogenator

Hydrogen production in a novel sonicated biological hydrogen reactor (SBHR) was investigated and compared with a continuous stirred tank reactor (CSTR). The two systems were operated at a hydraulic retention time (HRT) of 12 h and two organic loading rates (OLRs) of 21.4 and 32.1 g COD/L.d. The average hydrogen production rates per unit reactor volume for the conventional CSTR were 2.6 and 2.8 L/L.d, as compared with 4.8 and 5.6 L/L.d for SBHR, at the two OLRs, respectively. Hydrogen yields of 1.2 and 1.0 mol H₂/mol glucose were observed for the CSTR, respectively, while for the SBHR, the hydrogen yields were 2.1 and 1.9 mol H₂/mol glucose at the two OLRs, respectively. The hydrogen content in the SBHR’s headspace was higher than that in CSTR by 10% and 31% at OLRs of 21.4 and 32.1 g COD/L.d, respectively. Both glucose conversion efficiency and HAc/HBu ratio in the SBHR were higher than in the conventional CSTR at both OLRs. The biomass yield of about 0.32 g VSS/g COD observed in the CSTR and 0.23 g VSS/g COD in the SBHR substantiate the higher H₂ yield in the SBHR. DGGE analysis confirmed the specificity of the microbial hydrogen-producing culture in the SBHR, with two different hydrogen producers (Clostridium sp. and Citrobacter freundii) detected in the SBHR and not detected in the CSTR.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Enhanced biohydrogen production from tofu residue by acid/base pretreatment and sewage sludge addition

Anaerobic dark fermentation is considered a promising technology for clean energy production and waste reduction. In the present work, tofu residue and sewage sludge were utilized as substrates for fermentative hydrogen production. To increase the biodegradability, tofu residue was pretreated for 30 min in the presence of HCl and NaOH at various concentrations (0, 0.5, 1.0, and 2.0%), and then fermented by a thermophilic (60 °C) mixed culture. The solubility (SCOD/TCOD ratio) of the tofu residue increased from 4 to 30–40% after pretreatment, and the increased soluble constituents were mainly protein rather than carbohydrate compounds. However, in spite of a slight increase of carbohydrate solubility, the H₂ production performance was significantly enhanced by pretreatment, owing to the degradability of thermophilic cultures used on insoluble tofu residues. The limited H₂ yield of 0.30 mol H₂/mol hexose_added achieved in the raw tofu residue was increased 1.6- to 4-fold with the highest H₂ yield of 1.25 mol H₂/mol hexose_added at 1.0% HCl concentration. Carbohydrate degradation and the H₂ production rate also increased from 39 to 50–65% and 27 to 50–120 mL H₂/L/h, respectively. The role of pretreatment was not only to increase the biodegradability but also to suppress the activity of indigenous non H₂-producers such as lactic acid bacteria and propionic acid bacteria. When sewage sludge was added to acid pretreated (1.0% HCl) tofu residue as a co-substrate, the H₂ yield and H₂ production rate increased to 1.48 mol H₂/mol hexose_added and 161 mL H₂/L/h, respectively, which was attributed to the abundant minerals, vitamins, and metals contained in sewage sludge.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.