Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches

This study focuses on the exploitation of cheese whey as a source for hydrogen and methane, in a two-stage continuous process. Mesophilic fermentative hydrogen production from undiluted cheese whey was investigated at a hydraulic retention time (HRT) of 24 h. Alkalinity addition (NaHCO₃) or an automatic pH controller were used, to maintain the pH culture at a constant value of 5.2. The hydrogen production rate was 2.9 ± 0.2 L/Lreactor/d, while the yield of hydrogen produced was approximately 0.78 ± 0.05 mol H₂/mol glucose consumed, with alkalinity addition, while the respective values when using pH control were 1.9 ± 0.1 L/Lreactor/d and 0.61 ± 0.04 mol H₂/mol glucose consumed. The corresponding yields of hydrogen produced were 2.9 L of H₂/L cheese whey and 1.9 L of H₂/L cheese whey, respectively. The effluent from the hydrogenogenic reactor was further digested to biogas in a continuous mesophilic anaerobic bioreactor. The anaerobic digester was operated at an HRT of 20d and produced approximately 1 L CH₄/d, corresponding to a yield of 6.7 L CH₄/L of influent. The chemical oxygen demand (COD) elimination reached 95.3% demonstrating that cheese whey could be efficiently used for hydrogen and methane production, in a two-stage process.     

Semi-continuous biohydrogen production as an approach to generate electricity

In this work, a semi-continuous biological system was established to produce hydrogen and generate electricity by coupling the bioreactor to a fuel cell. Heat and acid pretreatments (at 35 and 55 °C) of a seed sludge used as inoculum were performed in order to increase hydrogen producers. Different initial glucose concentrations (IGC) were tested for heat pretreated inoculum at 35 °C to determine the optimum concentration of glucose that supported the highest hydrogen production. Results showed that the heat pretreated inoculums (35 °C) reached the highest hydrogen molar yield of 2.85 mol H₂/mol glucose (0.014 L/h), which corresponds to the acetic acid pathway. At the optimum IGC (10 g/L, 35 °C) the hydrogen molar yield was 3.6 mol H₂/mol glucose (0.023 L/h). The coupled bioreactor-fuel cell system yielded an output voltage of 1.06 V, power of 0.1 W (25 °C) and a current of 68 mA. The overall results suggest that high hydrogen molar yields can be obtained through the acetic acid pathway and that is feasible to generate electricity using hydrogen from the semi- continuous bioreactor.     

Enhanced methane and hydrogen production from municipal solid waste and agro-industrial by-products co-digested with crude glycerol

The effects of crude glycerol on the performance of single-stage anaerobic reactors treating different types of organic waste were examined. A reactor treating the organic fraction of municipal solid waste produced 1400 mL CH₄/d before the addition of glycerol and 2094 mL CH₄/d after the addition of glycerol. An enhanced methane production rate was also observed when a 1:4 mixture of olive mill wastewater and slaughterhouse wastewater was supplemented with crude glycerol. Specifically, by adding 1% v/v crude glycerol to the feed, the methane production rate increased from 479 mL/d to 1210 mL/d. The extra glycerol-COD added to the feed did not have a negative effect on the reactor performance in either case. Supplementation of the feed with crude glycerol also had a significant positive effect on anaerobic fermentation reactors. Hydrogen yield was 26 mmole H₂/g VS added and 15 mmole H₂/g VS added in a reactor treating the organic fraction of municipal solid waste and a 1:4 mixture of olive mill and slaughterhouse wastewater. The addition of crude glycerol to the feed enhanced hydrogen yield at 2.9 mmole H₂/g glycerol added and 0.7 mmole H₂/g glycerol added.     

Semi-continuous photo-fermentative H2 production by Rhodobacter sphaeroides: effect of decanting volume ratio

In this study, a semi-continuous operation of photo-fermentative H₂-producing reactor was attempted at various decanting volume ratios (DVR, decanting volume per day/total working volume, %), ranging 30-70%, using Rhodobacter sphaeroides KD131. H₂ production was not efficient with showing low H₂ yields of 0.2 and 0.5 mol H₂/mol succinate_added at 30% and 40% DVR, respectively. The low performance ascribed to the fact that over 70% of substrate electrons were diverted towards cell growth under these conditions. Meanwhile, cell growth was limited at DVR ? 50%; therefore, higher H₂ yields (>2.0 mol H₂/mol succinate_added) were observed. Both the highest H₂ yield of 3.7 mol H₂/mol succinate_added and production rate of 1494 mL H₂/L-reactor/d were achieved at 60% DVR. The content of soluble microbial products (SMPs) was measured, which accounted for 3-15% of substrate electrons. It was found that the largest (65-75%) portion of SMPs comprised low molecular-weight (<3 kDa).     

Biohydrogen from thermophilic co-fermentation of swine manure with fruit and vegetable waste: maximizing stable production without pH control

Hydrogen production by dark fermentation may suffer of inhibition or instability due to pH deviations from optimality. The co-fermentation of promptly degradable feedstock with alkali-rich materials, such as livestock wastes, may represent a feasible and easy to implement approach to avoid external adjustments of pH.Experiments were designed to investigate the effect of the mixing ratio of fruit-vegetable waste with swine manure with the aim of maximizing biohydrogen production while obtaining process stability through the endogenous alkalinity of manure.Fruit-vegetable/swine manure ratio of 35/65 and HRT of 2 d resulted to give the highest production rate of 3.27 ± 0.51 L_H2 L⁻¹ d⁻¹, with a corresponding hydrogen yield of 126 ± 22 mL_H2 g⁻¹_VS-added and H₂ content in the biogas of 42 ± 5%. At these operating conditions the process exhibited also one of the highest measured stability, with daily productions deviating for less than 14% from the average.     

Co-digestion of intermediate landfill leachate and sewage sludge as a method of leachate utilization

This study examines the co-digestion of intermediate landfill leachate and sewage sludge from a municipal wastewater treatment plant. Application of leachate as a co-fermentation component increased the concentrations of soluble organic compounds (expressed as total organic carbon), ammonium nitrogen, and alkalinity in the digester influents.The biogas yield obtained from the co-fermentation of a 20:1 sewage sludge: intermediate leachate mixture was 1.30 m³ per kg of removed volatile solids (VS), while that from a 10:1 mixture was 1.24 m³ per kg of removed VS. These values exceeded the biogas yield for the sludge alone by 13% and 8%, respectively. The leachate addition influenced the proportion of methane to a minor extent. Increased methane yields of 16.9% and 6.2% per kg of removed VS were found for the two sewage sluge:intermediate leachate mixtures, respectively.     

Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell

Hydrogen gas production from cellulose was investigated using an integrated hydrogen production process consisting of a dark fermentation reactor and microbial fuel cells (MFCs) as power sources for a microbial electrolysis cell (MEC). Two MFCs (each 25 mL) connected in series to an MEC (72 mL) produced a maximum of 0.43 V using fermentation effluent as a feed, achieving a hydrogen production rate from the MEC of 0.48 m³ H₂/m³/d (based on the MEC volume), and a yield of 33.2 mmol H₂/g COD removed in the MEC. The overall hydrogen production for the integrated system (fermentation, MFC and MEC) was increased by 41% compared with fermentation alone to 14.3 mmol H₂/g cellulose, with a total hydrogen production rate of 0.24 m³ H₂/m³/d and an overall energy recovery efficiency of 23% (based on cellulose removed) without the need for any external electrical energy input.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209 ml H₂/l medium, 1.97 mol H₂/mol glucose and 104.20 ml H₂/g CDW h⁻¹ were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H₂, and is another model microorganism for biohydrogen investigations.     

Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis

Hydrogen production was studied using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H₂ production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H₂ under immobilized state in agar. The externally added glucose enhanced H₂ production rates and total produced volume while shortened the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 mL/h/L and 238 mL/h/L at 37 °C and 40 °C, respectively. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis stage should be allowed for cells to recover H₂ production potential and cell viability before returning to H₂ production stage again.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 °C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H₂ mol⁻¹ glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter.     

Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study

In the present work the hydrogenesis in the anaerobic alkalithermophilic bacterium Thermobrachium celere was studied. The impact of several factors on hydrogen production during glucose fermentation was investigated in batch conditions. The optimal hydrogen production occurred at pH_67 °C 8.2 with phosphate buffer concentration of 50 mM. Hydrogen yield reached the highest value of 3.36 mol H₂/mol glucose when the partial pressure in the gas headspace was reduced. Supplementation of nitrogen sources and iron affected hydrogen production. Under optimized conditions, the maximum H₂ accumulation and H₂ production rate were estimated to be respectively 124.3 mmol H₂/l culture and 20.7 mmol H₂/l/h. Considering the efficient and rapid hydrogen evolution, and the ability to grow in extreme environments, T. celere might be a good candidate for biohydrogen production in open (non-sterile) bioprocess system.     

Fermentative hydrogen production from fresh leachate in batch and continuous bioreactors

This research for the first time investigated hydrogen production from the fresh leachate originated from municipal solid wastes. We found that fermentation of the leachate generated H₂ and was very much enhanced in the presence of extra phosphate in the batch reactor. The continuous expanded granular sludge bed (EGSB) reactor started to generate H₂ at day 20 and continued to 176 days with 120 mg/l of extra phosphate present. The highest chemical oxygen demand (COD) removal efficiency (66.9%) was achieved at liquid up-flow velocity of 3.7 m/h and hydraulic retention time of 12 h. Under proposed optimal operation conditions, the mean H₂ production rate reached up to 2155 ml/(l day). We also found that over 80% liquid metabolites were acetic acid and ethanol, suggesting the ethanol-type fermentation was dominant in the bioreactor. These findings indicate that the fresh leachate can be used as the source for continuous hydrogen production.     

Feasibility of hydrogen production from ripened fruits by a combined two-stage (dark/dark) fermentation system

Anaerobic fermentation for hydrogen (H₂) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H₂ production (4463.7 mL-H₂ L⁻¹-culture) with a maximum H₂ yield (2.2 mol H₂ mol⁻¹ glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H₂ L⁻¹-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H₂ conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.     

Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production

The application of a cell immobilization technique to a biofilm-based photobioreactor was developed to enhance its photo-hydrogen production rate and light conversion efficiency. Rhodopseudomonas palustris CQK 01 was initially attached to the surface of packed glass beads to form a biofilm in this experiment. Then, the biofilm photobioreactor (BPBR) was illuminated by light-emitting diodes with light wavelengths of 470, 590 and 630 nm and hydrogen was evolved with glucose being the sole carbon source. Under the illumination condition of 5000 lux illumination intensity and 590 nm wavelength, the BPBR showed good hydrogen production performance: the hydrogen production rate was 38.9 ml/l/h and light conversion efficiency was 56%, while the hydrogen yield was 0.2 mol H₂/mol glucose. Furthermore, results show that the highest hydrogen production rate and glucose removal rate were obtained when the glucose concentration is 0.12 M, the optimal pH 7 and optimal temperature of influent liquid 25 °C.     

Fermentative hydrogen production from Laminaria japonica and optimization of thermal pretreatment conditions

As a sustainable biofuel feedstock, marine algae have superior aspects to terrestrial biomass such as less energy and water requirement for cultivation, higher CO₂ capture capacity, and negligible lignin content. In this study, various marine algae were tested for fermentative hydrogen production (FHP). Among them, Laminaria japonica exhibited the best performance, showing the highest H₂ yield of 69.1 mL H₂/g COD_added. It was attributed to its high carbohydrate content and main constituents of polysaccharides, laminarin and alginate, which were found to posses higher H₂ production potential than agar and carrageenan. To enhance the H₂ production from L. japonica, thermal pretreatment was applied at various conditions. At 170 °C and 20 min, H₂ yield was maximized to 109.6 mL H₂/g COD_added. The experimental results suggested that marine algae, especially L. japonica, could be used for FHP, and future works would be focused on gaining more energy from the H₂ fermentation effluent.     

Effect of initial pH independent of operational pH on hydrogen fermentation of food waste

The effect of initial pH from 5.0 to 9.0 on H₂ fermentation of food waste was investigated. In this batch experiment, however, unlike previous studies for initial pH, operational pH was maintained at 5.0 by the addition of alkaline solution. Although the period for pH drop from the initial values to 5.0 was less than one-tenth of the entire fermentation, this short period significantly affected the H₂ production performance. At initial pH 6.0-9.0, successful H₂ yield of 1.3-1.9 mol H₂/mol hexose_added was achieved with a peak value at pH 8.0. The H₂ yield achieved at initial pH 8.0 was corresponded to the 8.13% of total energy content in the substrate. At initial pH 5.0, the smallest butyrate production, but the highest ethanol production was detected, indicating unfavorable conditions for H₂ production. There was no significant relationship between total required amount of alkaline solution and initial pH values.