Bio-hydrogen production from cheese whey powder (CWP) solution: Comparison of thermophilic and mesophilic dark fermentations

Cheese whey powder (CWP) solution was used as the raw material for hydrogen gas production by mesophilic (35 °C) and thermophilic (55 °C) dark fermentations at constant initial total sugar and bacteria concentrations. Thermophilic fermentation yielded higher cumulative hydrogen formation (CHF = 171 mL), higher hydrogen yield (111 mL H₂ g⁻¹ total sugar), and higher hydrogen formation rate (3.46 mL H₂ L⁻¹ h⁻¹) as compared to mesophilic fermentation. CHF in both cases were correlated with the Gompertz equation and the constants were determined. Despite the longer lag phase, thermophilic fermentation yielded higher specific H₂ formation rate (2.10 mL H₂ g⁻¹cells h⁻¹). Favorable results obtained in thermophilic fermentation were probably due to elimination of H₂ consuming bacteria at high temperatures and selection of fast hydrogen gas producers.     

Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation

Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were compared in batch dark fermentations under mesophilic (37 °C) and thermophilic (55 °C) conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 °C and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H₂ h⁻¹) was obtained with the acid-hydrolyzed and the lowest (1.12 ml H₂ h⁻¹) with the boiled wheat at 55 °C. The highest hydrogen gas yield (333 ml H₂ g⁻¹ total sugar or 2.40 mol H₂ mol⁻¹ glucose) and final total volatile fatty acid (TVFA) concentration (10.08 g L⁻¹) were also obtained with the acid-hydrolyzed wheat under thermophilic conditions (55 °C). Dark fermentation of acid-hydrolyzed ground wheat under thermophilic conditions (55 °C) was proven to be more beneficial as compared to mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.     

Impact of furfural on biological hydrogen production kinetics from synthetic lignocellulosic hydrolysate using mesophilic and thermophilic mixed cultures

The impact of furfural on hydrogen production and microbial growth kinetics was assessed using mixed anaerobic cultures at mesophilic and thermophilic conditions. Mesophilic experiments showed a hydrogen yield of 1.6 mol H₂/mol initial sugars at 1 g/L furfural which is a 45% enhancement from the control (0 g/L furfural) at a substrate-to-biomass ratio (S°/X°) of 4 gCOD/gVSS. On the other hand, thermophilic experiments showed no enhancement at 1 g/L furfural but rather a 53% decrease in hydrogen yield from its control. Furfural inhibition threshold limit was observed to be greater than 1 g/L for mesophilic experiments and less than 1 g/L for thermophilic experiments. In both cases, 4 g/L was the most recalcitrant furfural concentration, with propionate and lactate the most predominant soluble metabolites in the mesophilic and thermophilic experiments respectively. It was also noted that in the presence of furfural, hydrogen-producers in both mesophilic and thermophilic mixed cultures were inactivated as no hydrogen was produced until furfural was completely degraded irrespective of sugars degradation. This study also presents the kinetics of microbial growth and substrate degradation obtained using the Monod model on MATLAB^®, ignoring an inhibition term. IC₅₀ of the mesophilic and thermophilic experiments were 1.03 g/L and 0.5 g/L respectively indicating that the thermophilic hydrogen producers were more strongly affected by furfural than the mesophilic cultures.     

Optimization of biohydrogen production of palm oil mill effluent by ozone pretreatment

The biohydrogen (H₂) production in batch experiments under varying concentrations of raw and ozonated palm oil mill effluent (POME) of 5000–30,000 mg COD.L⁻¹, at initial pH 6, under mesophilic (37 °C), thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. Effects of ozone pretreatment, substrate concentration and fermentation temperature on H₂ production using mesophilic seed sludge was undertaken. The results demonstrated that H₂ can be produced from both raw and ozonated POME, and the amounts of H₂ production were directly increased as the POME concentrations were increased. H₂ was successfully produced under the mesophilic fermentation of ozonated POME, with maximum H₂ yield, and specific H₂ production rate of 182 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 6.2 mL.h⁻¹.g⁻¹ TVS (25,000 mg COD.L⁻¹), respectively. Thus, indicating that the ozone pretreatment could elevate on the biodegradability of major constituents of the POME, which significantly enhanced yields and rates of the H₂ production. H₂ production was not achieved under the thermophilic and extreme-thermophilic fermentation. In both fermentation temperatures with ozonated POME, the maximum H₂ yield was 62 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹) and 63 mL.g⁻¹ COD_removed (30,000 mg COD.L⁻¹), respectively. The highest efficiency of total and soluble COD removal was obtained at 44 and 37%, respectively following the mesophilic fermentation, of 24 and 25%, respectively under the thermophilic fermentation, of 32 and 20%, respectively under the extreme-thermophilic fermentation. The production of volatile fatty acids increased with an increased fermentation time and temperature in both raw and ozonated POME under all three fermentation temperatures. The accumulation of volatile fatty acids in the reactor content were mostly acetic and butyric acids. H₂ fermentation under the mesophilic condition of 37 °C was the better selection than that of the thermophilic and extreme-thermophilic fermentation.     

Bio-hydrogen production from tequila vinasses: Effect of detoxification with activated charcoal on dark fermentation performance

Hydrogen production from dark fermentation is a potential source of sustainable fuel when it is generated from waste. This study compared hydrogen production resulting from fermentation using raw and detoxified tequila vinasse. Vinasse was detoxified with granular activated charcoal, which was used to adsorb compounds that could inhibit the production of hydrogen by dark fermentation. In batch cultures detoxification of vinasse led to up to 20% higher maximum velocities of hydrogen production, a 5.4 h reduction in the lag phase and an 11% higher molar yield, compared to results obtained with raw vinasse. Losses of sugars after detoxification provoked that the specific hydrogen volumetric yields obtained with detoxified vinasse were 30–40% lower with 5 g COD/L and 15 g COD/L initial concentrations, compared to the ones obtained with raw vinasse. For an initial 30 g COD/L no differences in specific hydrogen yields were observed between raw or detoxified vinasse in batch fermentation. Continuous culture fermentation of vinasse showed hydrogen production rates between 1.32 ± 0.07 to 1.39 ± 0.14 NL H₂/L-d when extra nutrients were added, while a stable production of hydrogen through fermentation of detoxified vinasse could not be maintained despite nutrient addition. Production of hydrogen from vinasse diluted with water with no additional nutrients was assessed and rates close to 0.42 ± 0.02 NL H₂/L-d and hydrogen content close to 37% were obtained. Accumulation of lactic acid and a predominant production of butyric acid over acetic acid suggested that the fermentation dynamics of vinasse with no supplementary nutrients were especially susceptible to high substrate loading rates and prolonged hydraulic retention times.     

Cladophora sp. as a sustainable feedstock for dark fermentative biohydrogen production

Macroalgae are rich in carbohydrates which can be used as a promising substrate for fermentative biohydrogen production. In this study, Cladophora sp. biomass was fermented for biohydrogen production at various inoculum/substrate (I/S) ratios against a control of inoculum without substrate in laboratory-scale batch reactors. The biohydrogen production yield ranged from 40.8 to 54.7 ml H₂/g-VS, with the I/S ratio ranging from 0.0625 to 4. The results indicated that low I/S ratios caused the overloaded accumulation of metabolic products and a significant pH decrease, which negatively affected hydrogen production bacteria's metabolic activity, thus leading to the decrease of hydrogen fermentation efficiency. The overall results demonstrated that Cladophora sp. biomass is an efficient fermentation feedstock for biohydrogen production.     

Modeling of biohydrogen production using generalized multi-scale kinetic model: Impacts of fermentation conditions

This paper presents a new multi-scale kinetic model built upon the multi-stage growth Hypothesis for predicting biohydrogen production. The proposed model represents the significant factors affecting biohydrogen production using a sum of first-order kinetic terms with varying dynamics from slow to fast one. The current work investigates 52 case studies of biohydrogen production that show the double first-order kinetic model provides the best modeling fitness (R² > 0.99). This result suggests two prevalent pathways or microbial groups with distinct dynamics (i.e., fast and slow modes) in biohydrogen production. An increase in temperature (30 °C–43 °C) or substrate concentration (10 g/L to 40 g/L) and the use of simple substrates or mixed cultures can increase the fast-mode dominance up to 100% contribution. Model analysis suggests that the fast mode corresponds to the butyrate production pathway, the growth-associated hydrogen-producing activity, the easily-biodegradable substrates, or the quick hydrogen-producing groups.     

High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition

Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis was investigated under thermophilic condition. The optimum chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g·L⁻¹ and 6.5 with a hydrogen yield of 73 mL-H₂·gCOD⁻¹. The dark fermentation effluent consisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was 0.7 V with a hydrogen yield of 163 mL-H₂·gCOD⁻¹. The hydrogen yield of continuous two-stage dark fermentation and microbial electrolysis was 236 mL-H₂·gCOD⁻¹ with a hydrogen production rate of 7.81 L·L⁻¹·d⁻¹. The hydrogen yield was 3 times increased when compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the microbial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under thermophilic condition is a highly promising option to maximize the conversion of palm oil mill effluent into biohydrogen.     

Techno-economic evaluation of biohydrogen production from wastewater and agricultural waste

The world is facing serious climate change caused in part by human consumption of fossil fuel. Therefore, developing a clean and environmentally friendly energy resource is necessary given the depletion of fossil fuels, the preservation of the earth's ecosystem and self-preservation of human life. Biological hydrogen production, using dark fermentation is being developed as a promising alternative and renewable energy source, using biomass feedstock. In this study, beverage wastewater and agricultural waste were examined as substrates for dark fermentation to produce clean biohydrogen energy.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Application of nanotechnology in dark fermentation for enhanced biohydrogen production using inorganic nanoparticles

Fermentation is an important innovation by mankind and this process is used for converting organic substrate into useful products. Using natural conditions, specifically, light and dark conditions, photo-fermentation and dark fermentation techniques can be developed and operated under controlled conditions. Generally, products such as biofuels, bioactive compounds and enzymes have been produced using the dark fermentation method. However, the major requirement for today's industralized world is biofuels in its clean and pure forms. Biohydrogen is the most efficient and cleanest form of energy produced using dark fermentation of organic substrates. Nevertheless, the quantity of biohydrogen produced via dark fermentation is low. In order to increase the product quantity and quality, several internal and external stress or alterations are made to conventional fermentation conditions. In recent times, nanotechnology has been introduced to enhance the rate of dark fermentation. Nanoparticles (NPs), specifically, inorganic NPs such as silver, iron, titanium oxide and nickel have increased the production rate of biohydrogen. Therefore, the present review focuses on exploring the potential of nanotechnology in the dark fermentation of biohydrogen production, the mechanisms involved, substrates used and changes to be made to increase the production efficiency of dark fermentation.     

Comparative evaluation of the biohydrogen production potential of thermophilic microorganisms isolated from hot springs located in Izmir

Biohydrogen production with the utilization of thermophilic microorganisms is a green process contributing to the mitigation of fossil fuel dependency. This study reports an improved water-gas shift reaction (WGSR) methodology with isolation of thermophilic and anaerobic microorganisms from 5 different closely-located hot springs, employing enrichment and characterization techniques to evaluate their biohydrogen production capacities with 100% CO in the headspace. Enriched mixed and pure cultures were screened for biohydrogen production. An isolate from Doğanbey hot spring reached the highest H₂ yield of 0.43 mmol H₂/mmol CO with a 43% CO conversion. Using growth profiling and morphologically with SEM imaging, this isolate was characterized biochemically, and was identified as an isolated bacilli shaped bacteria capable of producing hydrogen through WGSR. This is the first study to report potential thermophilic hydrogen producers in selected hot springs in the Izmir region.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

Comparison of mesophilic and thermophilic anaerobic hydrogen production by hot spring enrichment culture

Anaerobic hydrogen producing mesophilic and thermophilic cultures were enriched and studied from an intermediate temperature (45 °C) hot spring sample. H₂ production yields at 37 °C and 55 °C were highest at the initial pH of 6.5 and 7.5, respectively. Optimum glucose, iron and nickel concentrations were 9 g/l, 25 mg/l and 25 mg/l both at 37 °C and 55 °C, respectively. The highest H₂ yields at 37 °C and 55 °C were 1.8 and 1.0 mol H₂/mol glucose, respectively, with the optimal pH, glucose concentration and iron addition. Hydrogen production from glucose at 55 °C and 37 °C was associated with ethanol- and acetate–butyrate type fermentations, respectively. Bacterial composition was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Clostridium species dominated at both temperatures and the microbial diversity decreased with increasing temperature. At 55 °C, Clostridium ramosum was the dominant organism.     

Co-digestion of corn (nejayote) and brewery wastewater at different ratios and pH conditions for biohydrogen production

The capacity of a heat-treated sludge (HTS) to produce hydrogen from the mono- and co-digestion of corn (NW, pH 13.1) and brewery (BW, pH 3.8) wastewater was evaluated. The co-digestion of NW and BW was conducted with ratios (NW/BW) from 40/60 to 80/20 (vol/vol) at pH 6 and under different initial pH values (from 5.8 to 12.3) according to the substrates mixtures. With the initial pH adjusted to 6, the highest production (302 mL) occurred for the mono-digestion of NW, but hydrogen was produced in all incubations. For incubations under variable pH values, the highest hydrogen production was obtained with the ratio 60/40 (270 mL), followed by the ratio 65/35 (260 mL) with pH values of 10.8 and 10.4, respectively. The initial pH influenced the kinetic parameters, especially on maximum production and lag phase. For the ratio 60/40, with an alkaline pH value (10.4), the lag phase was delayed up to 122 h, but the highest volume of hydrogen was obtained with this condition. The Clostridium genus, present in all samples, could be associated as the main responsible for hydrogen production. Besides, the presence of Burkholderia genus, previously related to hydrogen production, was identified as the main involved in the culture at pH 10.8.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Microbial community analysis of thermophilic mixed culture sludge for biohydrogen production from palm oil mill effluent

The microbial community structure of thermophilic mixed culture sludge used for biohydrogen production from palm oil mill effluent was analyzed by fluorescence in situ hybridization (FISH) and 16S rRNA gene clone library techniques. The hydrogen-producing bacteria were isolated and their ability to produce hydrogen was confirmed. The microbial community was dominated by Thermoanaerobacterium species (∼66%). The remaining microorganisms belonged to Clostridium and Desulfotomaculum spp. (∼28% and ∼6%, respectively). Three hydrogen-producing strains, namely HPB-1, HPB-2, and HPB-3, were isolated. 16S rRNA gene sequence analysis of HPB-1 and HPB-2 revealed a high similarity to Thermoanaerobacterium thermosaccharolyticum (98.6% and 99.0%, respectively). The Thermoanaerobacterium HPB-2 strain was a promising candidate for thermophilic fermentative hydrogen production with a hydrogen yield of 2.53 mol H₂ mol⁻¹hexose from organic waste and wastewater containing a mixture of hexose and pentose sugars. Thermoanaerobacterium species play a major role in thermophilic hydrogen production as confirmed both by molecular and cultivation-based analyses.     

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.     

Continuous biohydrogen production using cheese whey: Improving the hydrogen production rate

Due to the renewed interest in finding sustainable fuels or energy carriers, biohydrogen (Bio-H₂) from biomass is a promising alternative. Fermentative Bio-H₂ production was studied in a continuous stirred tank reactor (CSTR) operated during 65.6 d with cheese whey (CW) as substrate. Three hydraulic retention times (HRTs) were tested (10, 6 and 4 h) and the highest volumetric hydrogen production rate (VHPR) was attained with HRT of 6 h. Therefore, four organic loading rates (OLRs) at a fixed HRT of 6 h were tested thereafter, being: 92.4, 115.5, 138.6 and 184.4 g lactose/L/d. The highest VHPR (46.61 mmol H₂/L/h) and hydrogen molar yield (HMY) of 2.8 mol H₂/mol lactose were found at an OLR of 138.6 g lactose/L/d; a sharp fall in VHPR occurred at an OLR of 184.4 g lactose/L/d. Butyric, propionic and acetic acids were the main soluble metabolites found, with butyric-to-acetic ratios ranging from 1.0 to 2.4. Bacterial community was identified by partial sequence analysis of the 16S rRNA and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). The results showed that at HRT of 10 h and 6 h were dominated by the Clostridium genus. The VHPR attained in this study is the highest reported value for a CSTR system using CW as substrate with anaerobic sludge as inoculum and represents a 33-fold increase compared to a previous study. Thus, it was demonstrated that continuous fermentative Bio-H₂ production from CW can be significantly enhanced by an appropriate selection of parameters such as HRT and OLR. Enhancements in VHPR are significant because it is a critical parameter to determine the full-scale practical application of fermentation technologies that will be used for sustainable and clean energy generation.     

Enzymatic hydrolysis and anaerobic biological treatment of fish industry effluent: Evaluation of the mesophilic and thermophilic conditions

Enzymatic hydrolysis and anaerobic treatment of effluent similar to that generated in the fish processing industry were evaluated at 30 °C and 50 °C. Hydrolysis used lipase produced by fungus Penicillium simplicissimum in solid state fermentation with babassu cake as substrate, which has optimal activity at 50 °C. Hydrolysis kinetics was conducted with mixtures of effluent (containing 1500 mg oils and greases/L) and different lipase activities (0–0.67 U/ml of effluent), verifying that with 0.16 U/ml of effluent, 9.69 μmol/ml of free acids were produced after 4 h at 50 °C. Anaerobic biodegradation assays were conducted with effluent submitted to three different treatments: thermophilic (hydrolysis and anaerobic treatment at 50 °C), mesophilic (hydrolysis and anaerobic treatment at 30 °C) and hybrid (hydrolysis at 50 °C and anaerobic treatment at 30 °C). The best results (97.5% of chemical oxygen demand [COD] removal and 105.4 ml CH₄/g COD_removed) were obtained with the hybrid treatment in only 68 h. The thermophilic hydrolysis not only reduced the amount of enzyme and the hydrolysis time but also reduced the time and the cost of mesophilic anaerobic treatment, favoring the application of this treatment on an industrial scale.