Feasibility of biohydrogen production from Gelidium amansii

The feasibility of hydrogen production from red algae was investigated. Galactose, the main sugar monomer of red algae, was readily converted to hydrogen by dark fermentation. The maximum hydrogen production rate and yield of galactose were 2.46 L H₂/g VSS/d and 2.03 mol H₂/mol galactose_added, respectively, which were higher than those for glucose (0.914 L H₂/g VSS/d and 1.48 mol H₂/mol galactose_added). The distribution of soluble byproducts showed that H₂ production was the main pathway of galactose uptake. 5-HMF, the main byproduct of acid hydrolysis of red algae causes noncompetitive inhibition of H₂ fermentation. 1.37 g/L of 5-HMF decreased hydrogen production rate by 50% compared to the control. When red algae was hydrolyzed at 150 °C for 15 min and detoxified by activated carbon, 53.5 mL of H₂ was produced from 1 g of dry algae with a hydrogen production rate of 0.518 L H₂/g VSS/d. Red algae, cultivable on vast tracts of sea by sunlight without any nitrogen-based fertilizer, could be a suitable substrate for biohydrogen production.     

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.     

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.     

Specific inhibition of biohydrogen-producing Clostridium sp. after dilute-acid pretreatment of sunflower stalks

Dilute-acid pretreatments are commonly used to solubilize holocelluloses of lignocellulosic materials and represent a promising route to enhance biohydrogen production by dark fermentation. Besides the soluble sugars released, furan derivatives, such as furfural and 5-HMF, as well as phenolic compounds can accumulate in dilute-acid hydrolyzates and that may affect fermentative microbial populations. In this study, biohydrogen production from glucose (5 g VS L⁻¹) in batch tests was investigated in presence of increasing volumes (0% – control, 3.75%, 7.5%, 15% and 35% (v/v)) of dilute acid hydrolyzate generated from sunflower stalks (170 °C, 1 h, 4 g HCl/100 gTS). A sharp decrease of the hydrogen yield was observed from 2.04 mol H₂ mol⁻¹_{eq. hexose initial} in the control to 0 mol H₂ mol⁻¹_{eq. hexose initial} for volumes higher than 15% of added hydrolyzate. Although acetate and butyrate were the main end-products found in the control, ethanol and lactate accumulated accordingly with the increasing addition of hydrolyzate. A clear shift of dominant microbial populations from Clostridium sp. to Sporolactobacillus sp. was concomitantly observed, suggesting a specific inhibition of the biohydrogen-producing bacteria by adding increasing volumes of hydrolyzates.     

The effect of hydraulic retention time on thermophilic dark fermentative biohydrogen production in the continuously operated packed bed bioreactor

The main objective of the study is to investigate the effect of hydraulic retention times on continuous dark fermentative biohydrogen production in an up-flow packed bed reactor (UPBR) containing a novel microorganism immobilization material namely polyester fiber beads. The hydrogen producing dark fermentative microorganisms were obtained by heat-pretreatment of anaerobic sludge from the acidogenic phase of an anaerobic wastewater treatment plant. Glucose was the sole carbon source and the initial concentration was 15 ± 1 g/L throughout the continuous feeding. UPBR was operated under the thermophilic condition at T = 48 ± 2 °C and at varying HRTs between 2 h and 6 h. The hydrogen productivity of continuously operated UPBR increased with increasing HRT. Hydrogen production volume varied between 4331 and 6624 ml/d, volumetric hydrogen production rates (VHPR) were obtained as 3.09–4.73 L H₂/L day, and hydrogen production yields (HY) were 0.49 mol/mol glucose-0.89 mol/mol glucose depending on HRT. Maximum daily hydrogen volume (6624 ml/d), the yield (0.89 mol/mol glucose) and VHPR (4.73 L H₂/L day) were obtained at HRT = 6 h. The production rate and the yield decreased with increasing organic loading rate due to substrate inhibition.     

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.     

Characteristics of biohydrogen fermentation from various substrates

The characteristics of biohydrogen production from sucrose, slurry-type piggery waste and food waste under the effects of the reactor configurations and operational pHs (6 and 9) were examined by using heat-treated anaerobic sludge as a seed biomass. When sucrose was used in the batch test, the maximum hydrogen yield was 0.12–0.13 g COD (as H₂)/g COD (1.41–1.43 mol/mol hexose) at pH 6. In contrast, 0.10–0.11 g COD (as H₂)/g COD (1.12–1.21 mol/mol hexose) hydrogen yield was achieved from the reactor at pH 9. On the other hand, hydrogen production was not observed in the continuous sequencing batch mode fermenters fed with sucrose. Profile analysis at each cycle revealed hydrogen production at the initial operation periods but eventually only methane at 36 days. When slurry-type piggery waste was used as the substrate, the upflow elutriation-type fermenters produced methane but not hydrogen after 30 days operation. The fermentation intermediate profile showed that the hydrogen produced might have been consumed by homoacetogenic or propionate producing reactions, and eventually converted into methane by acetoclastic methanogens. The downflow leaching bed fermenters using food waste produced 0.013 L H₂/g volatile solids (VS) (0.0061 g COD (as H₂)/g COD) at pH 6 with 54% VS reduction whereas 0.0041 L H₂/g VS (0.0020 g COD (as H₂)/g COD) was produced at pH 9 with 86% VS reduction. The results show that the hydrogen produced should be released rapidly from the reactor before it can be consumed in other biochemical reactions, and substrates with high pH level (>9.0) can be used directly to produce hydrogen without needing to adjust the pH.     

Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration

Pre-heated elephant dung was used as inoculum to produce hydrogen from sugarcane bagasse (SCB) hydrolysate. SCB was hydrolyzed by H₂SO₄ or NaOH at various concentrations (0.25-5% volume) and reaction time of 60 min at 121 °C, 1.5 kg/cm² in the autoclave. The optimal condition for the pretreatment was obtained when SCB was hydrolyzed by H₂SO₄ at 1% volume which yielded 11.28 g/L of total sugar (1.46 g glucose/L; 9.10 g xylose/L; 0.72 g arabinose/L). The maximum hydrogen yield of 0.84 mol H₂/mol total sugar and the hydrogen production rate of 109.55 mL H₂/L day were obtained at the initial pH 6.5 and initial total sugar concentration 10 g/L. Hydrogen-producing bacterium (Clostridium pasteurianum) and non hydrogen-producing bacterium (Flavobacterium sp.) were dominating species in the elephant dung and in hydrogen fermentation broth. Sporolactobacillus sp. was found to be responsible for a low hydrogen yield obtained.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

H2-rich biogas recirculation prevents hydrogen supersaturation and enhances hydrogen production by Thermotoga neapolitana cf. capnolactica

This study focused on the supersaturation of hydrogen in the liquid phase (H_2aq) and its inhibitory effect on dark fermentation by Thermotoga neapolitana cf. capnolactica by increasing the agitation (from 100 to 500 rpm) and recirculating H₂-rich biogas (GaR). At low cell concentrations, both 500 rpm and GaR reduced the H_2aq from 30.1 (±4.4) mL/L to the lowest values of 7.4 (±0.7) mL/L and 7.2 (±1.2) mL/L, respectively. However, at high cell concentrations (0.79 g CDW/L), the addition of GaR at 300 rpm was more efficient and increased the hydrogen production rate by 271%, compared to a 136% increase when raising the agitation to 500 rpm instead. While H_2aq primarily affected the dark fermentation rate, GaR concomitantly increased the hydrogen yield up to 3.5 mol H₂/mol glucose. Hence, H_2aq supersaturation highly depends on the systems gas-liquid mass transfer and strongly inhibits dark fermentation.     

Enhanced photo-hydrogen production of Rhodopseudomonas faecalis RLD-53 by EDTA addition

This study investigated the effect of EDTA concentration in medium on the growth, hydrogen production and nitrogenase activity of Rhodopseudomonas faecalis RLD-53 by batch cultures. Experimental results indicated that bacterial growth and hydrogen production were strongly inhibited with EDTA concentration increasing to 0.6–0.7 g/L. However, the lag time of hydrogen production and the trends of biomass at EDTA concentration of 0–0.5 g/L were similar. The maximum cumulative hydrogen volume of 3325 ml H₂/L culture, hydrogen production rate of 27.6 ml H₂/L/h and hydrogen yield of 2.97 mol H₂/mol acetate were obtained when EDTA concentration was at 0.3 g/L, and the maximum OD₆₆₀ attained 3.83. And the nitrogenase activity also reached a maximum value of 1331.9 μl-C₂H₄/h/mg dry weight in the medium containing Fe²⁺ and EDTA. These results showed that a proper concentration of EDTA can promote the availability of iron, thereby further enhancing the activity of nitrogenase and photo-hydrogen production.     

Bioenergy production from glycerol in hydrogen producing bioreactors (HPBs) and microbial fuel cells (MFCs)

The supply of glycerol has increased substantially in recent years as a by-product of biodiesel production. To explore the value of glycerol for further application, the conversion of glycerol to bioenergy (hydrogen and electricity) was investigated using Hydrogen Producing Bioreactors (HPBs) and Microbial Fuel Cells (MFCs). Pure-glycerol and the glycerol from biodiesel waste stream were compared as the substrates for bioenergy production. In terms of hydrogen production, the yields of hydrogen and 1,3-propanediol at a pure-glycerol concentration of 3 g/L were 0.20 mol/mol glycerol and 0.46 mol/glycerol, respectively. With glucose as the co-metabolism substrate at the ratio of 3:1 (glycerol:glucose), the yields of hydrogen and 1,3-propanediol from glycerol significantly increased to 0.37 mol/mol glycerol and 0.65 mol/glycerol, respectively. The glycerol from biodiesel waste stream had good hydrogen yields (0.17-0.18 mol H₂/mole glycerol), which was comparable with the pure-glycerol. In terms of power generation in MFCs, pure-glycerol was examined at concentrations of 0.5-5 g/L with the highest power density of 4579 mW/m³ obtained at a concentration of 2 g/L. The power densities from the biodiesel waste glycerol were 1614-2324 mW/m³, which were likely caused by the adverse effects of impurities on electrode materials. An economic analysis indicates that with the annual waste stream of 70 million gallons of glycerol, the expected values generated from HPBs and MFCs were $311 and $98 million, respectively.     

Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract

The aim of the present study was to assess the influence of substrate concentration on the fermentative hydrogen production from sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time (HRT) of 12 h and carbohydrate concentrations ranging from 9.89 to 20.99 g/L, in glucose equivalents. The maximum hydrogen production rate and yield were obtained at the concentration of 17.50 g carbohydrates/L and were 2.93 ± 0.09 L H₂/L reactor/d and 0.74 ± 0.02 mol H₂/mol glucose consumed, corresponding to 8.81 ± 0.02 L H₂/kg sweet sorghum, respectively. The main metabolic product at all steady states was butyric acid, while ethanol production was high at high substrate concentrations. The experiments showed that hydrogen productivity depends significantly on the initial carbohydrate concentration, which also influences the distribution of the metabolic products.     

Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR)

Continuous biological hydrogen production from sweet sorghum syrup by mixed cultures was investigated by using anaerobic sequencing batch reactor (ASBR). The ASBR was conducted based on the optimum condition obtained from batch experiment i.e. 25 g/L of total sugar concentration, 1.45 g/L of FeSO₄ and pH of 5.0. Feasibility of continuous hydrogen fermentation in ASBR operation at room temperature (30 ± 3 °C) with different hydraulic retention time (HRT) of 96, 48, 24 and 12 hr and cycle periods consisting of filling (20 min), settling (20 min), and decanting (20 min) phases was analyzed. Results showed that hydrogen content decreased with a reduction in HRT i.e. from 42.93% (96 hr HRT) to 21.06% (12 hr HRT). Decrease in HRT resulted in a decrease of solvents produced which was from 10.77 to 2.67 mg/L for acetone and 78.25 mg/L to zero for butanol at HRT of 96 hr-12 hr, respectively. HRT of 24 hr was the optimum condition for ASBR operation indicated by the maximum hydrogen yield of 0.68 mol H₂/mol hexose. The microbial determination in DGGE analysis indicated that the well-known hydrogen producers Clostridia species were dominant in the reacting step. The presence of Sporolactobacillus sp. which could excrete the bacteriocins causing the adverse effect on hydrogen-producing bacteria might responsible for the low hydrogen content obtained.     

Improved photo-fermentative hydrogen production by biofilm reactor with optimizing carriers and acetate concentration

To enhance photo-fermentative hydrogen production (PFHP), biofilm reactor (BR) was employed as an ideal strategy with optimization on key factors of acetate concentration and carriers in this work. Optimal conditions for hydrogen production were acetate concentration of 4 g/L and carriers (silicon sheet) of 10 cm × 1 cm at amount of 1 piece. Biofilm formed on silicon sheet strongly improved hydrogen production compared with control reactor (CR). Cumulative hydrogen volume was enhanced about 20% from 2850 ± 130 mL/L of CR to 3349 ± 153 mL/L of BR and hydrogen yield was increased 20% from 2.61 ± 0.13 mol H₂/mol acetate of CR to 3.06 ± 0.15 mol H₂/mol acetate of BR at 4 g/L acetate. Protein and deoxyribonucleic acid (DNA) were important components to form the biofilm and they occupied 90% of extracellular polymeric substances (EPS). In particular, DNA, nearly 50% content of EPS, likely indicated a substantial contribution to biofilm formation and bacterial communication. Moreover, it suggested biofilm could regulate free cells to decline EPS secretion for improved hydrogen production. This work indicates BR could be a promising and economic strategy to enhance hydrogen production by photo-fermentation.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

Converting waste molasses liquor into biohydrogen via dark fermentation using a continuous bioreactor

This study investigated the effects of substrate concentration, HRT (hydraulic retention time), and pre-treatment of the substrate molasses on biohydrogen production from waste molasses (condensed molasses fermentation solubles, CMS) with a CSTR (continuously-stirred tank reactor). First, the hydrogen production was performed with various CMS concentrations (40–90 g COD/L, total sugar 8.7–22.6 g/L) with 6 h HRT. The results show that the maximal hydrogen production rate (HPR) occurred at 80 g COD/L substrate (19.8 g ToSu/L, ToSu: Total Sugar), obtaining an HPR of 0.417 mol/L/d. However, maximum hydrogen yield (HY) of 1.44 mol H₂/mol hexose and overall hydrogen production efficiency (HPE) of 25.6% were achieved with a CMS concentration of 70 g COD/L (17.3 g ToSu/L). The substrate inhibition occurred when CMS concentration was increased to 90 g COD/L (22.6 g ToSu/L). Furthermore, it was observed that the optimal HPR, HY, and HPE all occurred at HRT 6 h. Operating at a lower HRT of 4 h decreased the hydrogen production performance because of lower substrate utilization efficiency. The employment of pre-heating treatment (60 °C for 1 h) of the substrate could markedly enhance the fermentation performance. With 6 h HRT and substrate pre-heating treatment, the HPE raised to 29.9%, which is 18% higher than that obtained without thermal pretreatment.