Fermentative hydrogen production from macro-algae Laminaria japonica using anaerobic mixed bacteria

In this study, one macro-alga (Laminaria japonica) was used for fermentative hydrogen production by anaerobic mixed bacteria. The saccharification efficiency and hydrogen production by L. japonica with four different pretreatment methods, including heat, acid, alkaline and ultrasonic treatment, were investigated. The results showed that the saccharification efficiency from L. japonica that was pretreated with acid was the highest among the four methods. The saccharification efficiency for the total reducing sugars in the acid-pretreated L. japonica was 350.54 ± 19.89 mg/g (mean ± S.E.). The cumulative hydrogen production was 66.68 ± 5.68 mL/g from the heat-pretreated L. japonica, whereas that of L. japonica that was subjected to acid, alkaline, and ultrasonic pretreatment and the control was 43.65 ± 6.87 mL/g, 15.00 ± 3.89 mL/g, 23.56 ± 4.56 mL/g and 10.00 ± 1.21 mL/g, respectively. In addition, the effects of substrate concentration and initial pH on hydrogen production from heat-pretreated L. japonica were also analyzed. The results showed that the maximum hydrogen production was 83.45 ± 6.96 mL/g with a hydrogen concentration of approximately 28.4% from heat-pretreated L. japonica when the initial pH and substrate concentration were determined to be 6.0 and 2%, respectively. Heat pretreatment was the most effective method for increasing fermentative hydrogen production when L. japonica was used as the only substrate.     

Optimization of combined (acid + thermal) pretreatment for fermentative hydrogen production from Laminaria japonica using response surface methodology (RSM)

In the present work, a combined (acid + thermal) pretreatment was applied for the enhanced fermentative H₂ production (FHP) from Laminaria japonica. Various pretreatment conditions including HCl concentrations, heating temperatures, and reaction times were optimized via response surface methodology (RSM) with a Box-Behnken design (BBD). Through regression analysis, it was found that H₂ yield was well fitted by a quadratic polynomial equation (R² = 0.97), and the HCl concentration was the most significant factor influencing FHP. The desirable pretreatment conditions found were HCl concentration of 4.8%, temperature of 93 °C, and reaction time of 23 min, under which H₂ yield reached to 159.6 mL H₂/g dry cell weight (dcw). The main organic acids produced were acetic and butyric acids that related closely with H₂ production. The concentration of hydroxymethylfurfural (HMF), a byproduct formed during the pretreatment process, showed an inverse relationship with H₂ yield, indicating that pretreatment conditions for the H₂ production from L. japonica were successfully optimized, by increasing the hydrolysis rate of the feedstock and also reducing the formation of HMF.     

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.     

Inhibitory effect of chloroform on fermentative hydrogen and methane production from lipid-extracted microalgae

To improve the sustainability of microalgae as a bioenergy feedstock, lipid-extracted microalgae (LEM) are often further treated by anaerobic digestion (AD). However, the residual chloroform used for extracting lipids as a solvent could inhibit this process, an aspect that has not been studied to date. In this study, the inhibitory effect of chloroform on H₂ and CH₄ production was investigated by performing batch tests. To prepare the feedstock, Chlorella vulgaris was ultrasonicated and the supernatant was discarded after centrifugation. In case of H₂ production, it was found that the H₂ yield fell to almost half that of the control (15.6 mL H₂/g COD_added) at 100 mg CHCl₃/L. The reason for the decrease of the H₂ yield with the increase of chloroform level was due to the change of metabolites from acetate and butyrate to lactate via a non-hydrogenic reaction. In comparison with H₂ production, a much more severe inhibitory effect of chloroform on CH₄ production was observed. The inhibitor concentration (IC_{30, 60, and 90}) on H₂ production was 138, 319, and 622 mg CHCl₃/L, respectively, while concentrations of 15, 37, and 86 mg CHCl₃/L were obtained on CH₄ production. When the chloroform concentration was ≥25 mg/L on CH₄ production, more than 2 g COD/L of organic acids remained, resulting in a decrease of CH₄ yield. These findings indicate that the residual chloroform in LEM should be seriously considered to prevent possible microbial inhibition when designing a process for additional energy recovery from microalgae via AD.     

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.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

A pilot-scale study of biohydrogen production from distillery effluent using defined bacterial co-culture

We evaluated the feasibility of improving the scale of hydrogen (H₂) production from sugar cane distillery effluent using co-cultures of Citrobacter freundii 01, Enterobacter aerogenes E10 and Rhodopseudomonas palustris P2 at 100 m³ scale. The culture conditions at 100 ml and 2 L scales were optimized in minimal medium and we observed that the co-culture of the above three strains enhanced H₂ productivity significantly. Results at the 100 m³ scale revealed a maximum of 21.38 kg of H₂, corresponding to 10692.6 mol, which was obtained through batch method at 40 h from reducing sugar (3862.3 mol) as glucose. The average yield of H₂ was 2.76 mol mol⁻¹ glucose, and the rate of H₂ production was estimated as 0.53 kg/100 m³/h. Our results demonstrate the utility of distillery effluent as a source of clean alternative energy and provide insights into treatment for industrial exploitation.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

Biohydrogen production from ricebran using Clostridium saccharoperbutylacetonicum N1-4

Treated ricebran hydrolysate was fermented anaerobically using Clostridium saccharoperbutylacetonicum N1-4 at an initial pH of 6 ± 0.2 and an operating temperature of 30 °C for production of hydrogen. The effects of different pretreatment methods on the liberation of sugar from 100 g of ricebran per litre of medium (distilled water) were investigated. In addition, the effects of the pretreatment method on ricebran hydrolysates of different initial ricebran concentrations on liberated sugar as well as the effects of the initial inoculum concentration, ricebran (substrate) concentration, and FeSO₄·7H₂O concentration on the yield as well as the productivity of hydrogen were investigated. The combination of enzymatic hydrolysis and a boiling pretreatment method produced the most fermentable sugar, 29.03 ± 0.0 g/L from 100 g of ricebran per litre of medium (distilled water), while the amount of sugar liberated by ricebran hydrolysates of different initial ricebran concentrations upon pretreatment monotonically increased with the initial ricebran concentration. The increment in substrate, inoculum, and FeSO₄·7H₂O concentrations had a significantly positive effect (p < 0.05) on both the yield and productivity of hydrogen. The maximum hydrogen gas yield (Y_P/S) and productivity of 3.37 mol-H₂ per mol-sugar consumed and 7.58 mmol/(L h), respectively, were obtained from ricebran hydrolysate with a 100 g/L ricebran concentration (equivalent to 28.59 ± 1.27 g sugar/L). In other experiments, 0.03 g/L FeSO₄·7H₂O and 1.5 g/L inoculum resulted in the best hydrogen gas yield and productivity from ricebran hydrolysates.     

Synthesis gas production from dry reforming of methane over Ce0.75Zr0.25O2-supported Ru catalysts

Ce_0.75Zr_0.25O₂ solid solution supported Ru catalysts were prepared and tested for CH₄–CO₂ reforming. The effect of Ru content on the properties of the catalysts was investigated by means of N₂ adsorption–desorption, H₂-TPR/MS, XRD, XPS, CO chemisorption and H₂-TPD/MS. It was found that the highly dispersed Ru species favored the interaction between Ru and Ce_0.75Zr_0.25O₂. The reduced Ce_0.75Zr_0.25O₂ was able to store hydrogen, while Ru promoted the reduction of Ce_0.75Zr_0.25O₂. Under the identical conditions, the CH₄ and CO₂ conversions of the catalysts increased with the increase of Ru content, however, the turnover frequencies of CH₄ and CO₂ were higher for the catalysts with lower Ru contents, which may be resulted from the strong interaction between Ru and Ce_0.75Zr_0.25O₂. The Ru catalyst exhibited good stability and excellent resistance to carbon deposition. Remarkably, zirconium and cerium hydrides were detected in the used catalyst, which may participate in the elimination of the carbon deposit. Apart from the nature of metallic Ru and the redox property of Ce_0.75Zr_0.25O₂, we suggest that the excellent resistance of the catalyst to carbon deposition is also attributed to the hydrogen storage of the reduced Ce_0.75Zr_0.25O₂.     

Stimulatory effect of ascorbate,the alternative electron donor of photosystem II,on the hydrogen production of sulphur-deprived Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700⁺ and hydrogen evolution.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

Preparation and characterization of PPSU/PBNPI blend membrane for hydrogen separation

Novel polymer blend membranes of poly(bisphenol A-co-4-nitrophthalic anhydride-co-1,3-phenylenediamine) (PBNPI) and polyphenylsulfone (PPSU) in different weight ratios were prepared by a solution casting technique with N-methyl-2-pyrrolidone (NMP) as solvent. The effects of blend polymer composition on the membrane structure and the H₂, CO₂ and CH₄ separation performance were investigated. The membranes appear macroscopically miscible but microscopically immiscible based on thin-film X-ray diffraction investigations. A remarkably and continuously enhanced permeability has been achieved for these gases with increasing PPSU content from 0 to 50%. The highest pure H₂, CO₂ and CH₄ permeability are, respectively, equal to 40.4, 34.1 and 8.0 barrer.     

Engineering strategies for the enhanced photo-H2 production using effluents of dark fermentation processes as substrate

The major obstacle of combining dark and photo fermentation for high-yield biohydrogen production is substrate inhibition while using dark fermentation effluent as the sole substrate. To solve this problem, the dark fermentation broth was diluted with different dilution ratio to improve photo-H₂ production performance of an indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5. The best photo-H₂ production performance occurred at a dilution ratio of 1:2, giving a highest overall H₂ production rate of 10.72 ml/l/h and a higher overall H₂ yield of 6.14 mol H₂/mol sucrose. The maximum H₂ content was about 88.1% during the dilution ratio of 1:2. The photo-H₂ production performance was further improved by supplying yeast extract and glutamic acid as the nutrient. The results indicate that the overall H₂ production rate and H₂ yield increased to 17.02 ml/l/h and 10.25 mol H₂/mol sucrose, respectively. Using a novel solar-energy-excited optical fiber photobioreactor (SEEOFP) with supplementing tungsten filament lamp (TL) irradiation, the overall H₂ production rate was improved to 17.86 ml/l/h. Meanwhile, the power consumption by combining SEEOFP and TL was about 37.1% lower than using TL alone. This study demonstrates that using optimal light sources and proper dilution of dark fermentation effluent, the performance of photo-H₂ production can be markedly enhanced along with a reduction of power consumption.     

Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation

Chlamydomonas reinhardtii UTEX 90 accumulated 1.45 g dry cell weight and 0.77 g starch/L during photosynthetic growth using TAP media at 25 ^°C${}^{°}\mathrm{C}$ in presence of 2% _CO2${\mathrm{CO}}_2$ for 3 days. C. reinhardtii biomass was concentrated and then converted into hydrogen and organic acids by anaerobic fermentation with Clostridium butyricum. Organic acids in the fermentate of algal biomass were consecutively photo-dissimilated to hydrogen by Rhodobacter sphaeroides KD131. In the concentrated algal biomass 52% of the starch was hydrolyzed to 37.1 mmol _H2${\mathrm{H}}_2$/L-concentrated algal biomass and 13.6, 25.5, 7.4 and 493 mM of formate, acetate, propionate, and butyrate, respectively by C. butyricum. R. sphaeroides KD131 evolved 5.72 mmol _H2${\mathrm{H}}_2$ per ml-fermentate of algal biomass under illumination of 8 klux at 30 ^°C${}^{°}\mathrm{C}$. Only 80% of the organic acids, mainly butyrate, were hydrolyzed during photo-incubation. During anaerobic conversion, 2.58 mol _H2/mol${\mathrm{H}}_2/\mathrm{mol}$ starch–glucose was evolved using C. butyricum and then 5.72 mol _H2/L${\mathrm{H}}_2/\mathrm{L}$-anaerobic fermentate was produced by R. sphaeroides KD131. Thus, the two-step conversion process produced 8.30 mol _H2${\mathrm{H}}_2$ from 1 mol starch–glucose equivalent algal biomass via organic acids.     

State estimation of a batch hydrogen production process using the photosynthetic bacteria Rhodobacter capsulatus

This paper addresses the problem of estimating the states of an anaerobic photosynthetic process used for biohydrogen production by the photosynthetic bacterium Rhodobacter capsulatus. The process is described by a non-linear, time-discrete model and the state estimation is solved using an observer based on the Moving-Horizon State Estimation Method (MHSE). This approach is based on the minimization of a criterion (a non-linear function), in this case, the difference between the estimated output and the measured output of the system over a considered time horizon, where the solution is computed by using a numerical interval method. The observer was successfully applied to hydrogen production by R. capsulatus strain B10 in a batch process.     

A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OL

Caldicellulosiruptor owensensis strain OL^T (DSM 13100) is an obligately anaerobic, extreme thermophilic bacterium that is capable of utilizing a broad range of carbohydrates and producing H₂ as a metabolic by-product. The performance of C. owensensis on glucose and xylose was analyzed in lab-scale bioreactors to assess its potential use in biohydrogen production. Acetate, H₂, and CO₂ were the main end products during exponential growth of the organism on either sugar. Lactate production was triggered during the transition into the stationary phase and was associated with an increase in the levels of specific l-lactate dehydrogenase activity. In addition, minor amounts of ethanol and propionate could be detected. H₂ and acetate yields were lower on xylose than on glucose, marking an opposite trend to biomass and lactate yields. The influence of elevated H₂ partial pressure on product distribution was more dramatic in xylose-fermenting cultures. Replacement of yeast extract in the medium with a standard vitamins solution improved H₂ yield on both sugars, where it reached 100% of the theoretical maximum, i.e. 4 mol per mol hexose, on glucose. By using the defined medium, both the maximum specific growth rate and the maximum volumetric H₂ production rate of C. owensensis increased significantly on glucose and almost doubled on xylose. Screening other sugars besides glucose and xylose revealed a clear sugar-dependent product-distribution pattern and a direct correlation between biomass and lactate yields, which might be explained considering energy metabolism of the cells. The organism is proposed as a new candidate for biohydrogen production at high yields.