Single stage hydrogen production from cellulose through photo-fermentation by a co-culture of Cellulomonas fimi and Rhodopseudomonas palustris

Biohydrogen production from cellulose by a bacterial co-culture is a potentially promising approach for producing bioenergy from a low cost substrate. The use of a cellulolytic bacterium, Cellulomonas fimi, permits cellulose conversion and the in situ production of substrate for growth and hydrogen production by the photosynthetic bacterium Rhodopseudomonas palustris. Response surface methodology (RSM) with a Box-Behnken design (BBD) was used to examine variations in the key parameters: substrate (cellulose) concentration, yeast extract concentration and the microorganism ratio (Rps. palustris/C. fimi). For the co-culture of R. palustris and C. fimi the highest hydrogen production (44 mmol H₂/L) was achieved at the highest substrate concentration (5 g/L); however, the highest hydrogen yield (3.84 mol H₂/mol glucose equivalent) was observed at the lowest cellulose concentration and highest microorganism ratio. High COD removal efficiencies, over 70%, were achieved over a wide range of conditions and were positively affected by the concentration of yeast extract.     

Photo-fermentative hydrogen production by Rhodopseudomonas palustris CGA009 in the presence of inhibitory compounds

This study investigated Rhodopseudomonas palustris CGA009 biohydrogen production from compounds commonly found in lignocellulosic steam explosion hydrolysate, by examining the effect of individual inhibitory phenolic and furan compounds found in hydrolysates, under photo-fermentative anaerobic conditions. Since lignocellulose is often converted into ethanol via yeast-mediated fermentation, the tolerance of R. palustris CGA009 towards ethanol inhibition was also tested at a concentration range of 0.25–14% (v/v) under anaerobic photo-fermentative conditions. Hydrogen production was enhanced by compounds such as syringaldehyde (0.03 g/L), which accumulated total hydrogen of 960 mL over the cultivation period. In contrast, a reduction in hydrogen production of 1.4 fold was observed in vanillin-containing solutions (0.43 g/L), which obtained accumulated total hydrogen of 576 mL. Increasing ethanol concentrations reduced hydrogen production, but cell growth was not affected up to 1% (v/v), a fairly low concentration. R. palustris CGA009 can tolerate comparatively high concentrations of phenolic compounds, suggesting its use for lignocellulose hydrolysate detoxification and hydrogen production.     

Photoproduction of hydrogen by dye-sensitized systems

Visible light-induced production of hydrogen has been investigated in five different systems. These are: safranine O/EDTA, safranine T/EDTA, proflavine/EDTA, acridine orange/EDTA, and acridine yellow/EDTA, with and without added K₂PtCl₆. In the two safranine systems photoproduction of hydrogen was observed even in the absence of a Pt catalyst. Also, the addition of an electron mediator such as methyl viologen was found not necessary. Acridine yellow/EDTA/K₂PtCl₆ has been shown to be the best system examined, which upon addition of Triton X-100, showed further enhancement of the rate of hydrogen evolution.     

Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19

A newly isolated Citrobacter sp. Y19 for CO-dependent H₂ production was studied for its capability of fermentative H₂ production in batch cultivation. When glucose was used as carbon source, the pH of the culture medium significantly decreased as fermentation proceeded and H₂ production was seriously inhibited. The use of fortified phosphate at 60–180 mM alleviated this inhibition. By increasing culture temperatures (25–36°C), faster cell growth and higher initial H₂ production rates were observed but final H₂ production and yield were almost constant irrespective of temperature. Optimal specific H₂ production activity was observed at 36°C and pH 6–7. The increase of glucose concentration (1–20 g/l) in the culture medium resulted in higher H₂ production, but the yield of H₂ production (mol H₂/mol glucose) gradually decreased with increasing glucose concentration. Carbon mass balance showed that, in addition to cell mass, ethanol, acetate and CO₂ were the major fermentation products and comprised more than 70% of the carbon consumed. The maximal H₂ yield and H₂ production rate were estimated to be 2.49 molH₂/mol glucose and 32.3 mmolH₂/gcellh, respectively. The overall performance of Y19 in fermentative H₂ production is quite similar to that of most H₂-producing bacteria previously studied, especially to that of Rhodopseudomonas palustris P4, and this indicates that the attempt to find an outstanding bacterial strain for fermentative H₂ production might be very difficult if not impossible.     

Yeast and enzymatic hydrolysis in converting Chlorella biomass into hydrogen gas by Rhodobacter sp. and Rhodopseudomonas palustris

Enhanced hydrogen evolution was pursued in this work. Rhodobacter sp. (Rb) and Rhodopseudomonas palustris (Rp), single or mixed were used to extract hydrogen molecules from Chlorella fusca biomass. To elevate their fermentable contents, Chlorella was grown at nitrogen and/or phosphorus deprivation. Besides, cellulase and/or macerozyme, Triton X100 or sonicated yeast were applied for further biohydrogen fermentation. Utilizing hydrolysates of mineral deprived Chlorella cultures, Rb exhibited relatively higher cumulative hydrogen (4200 ml L^?1) than Rp (2500 ml L^?1) while mixed cultures attained significantly higher levels (4700 ml L^?1). Triton or enzymes significantly enhanced hydrogen evolution, with more effectiveness of macerozyme than cellulase. A novel use of sonicated yeast, as enzymes pool, induced the highest significant collective H₂ (up to 47 times that of microalgal supernatant). Sonicated yeast induced a remarkable hydrolysis of algae, as inferred from increased reducing sugars. However, hydrogen evolution efficiency exhibited poor proportionality with reducing sugars, indicating fermentation of other metabolites.     

Hydrogen production by immobilized Rhodopseudomonas palustris in packed or fluidized bed photobioreactor systems

The production of biohydrogen via photofermentation has been shown to have a low environmental impact and can often be integrated into wastewater treatment systems. However, currently, photofermentation has low production rates in comparison to industrial hydrogen production processes, and therefore requires improvement. One route for enhancing hydrogen productivity is the development of improved photobioreactor (PBR) systems. The aim of this study was to compare the hydrogen productivity of Rhodopseudomonas palustris under planktonic, and immobilized cell conditions, with the reactor operating either as a packed bed or a fluidized bed. The fluidized bed PBR achieved a maximum specific hydrogen production rate and substrate conversion efficiency of 15.74 ± 2.2 mL/g/h and 43% respectively, outperforming the conventional planktonic culture and the packed bed PBR. This work demonstrates a significant improvement in productivity over planktonic photofermentation, as well as demonstrating the use of immobilized cells under reactor conditions not usually associated with photosynthetic systems.     

Characteristics of a phototrophic sludge producing hydrogen from acetate and butyrate

A mixed phototrophic sludge was enriched from the sediment of a local river for continuous hydrogen production from acetate and butyrate in a complete-mix reactor. At pH 7.0–7.5 and , the optimal hydrogen production rate at 48 h of hydraulic retention time (HRT) for 150 days of steady-state operation averaged with of biomass. The sludge yield averaged -VSS/g-COD. Results of batch experiments showed an optimal pH of 8.5 and an optimal concentration of 2 mM for hydrogen production. At 10 mM, severely inhibited the hydrogen production. Three of the five OTUs classified from 26 clones developed from the seed sludge were phototrophs, based on phylogenetic analysis. Among them, OTU LA15, which is closely related to Rhodobacter sp., was most likely responsible to the hydrogen production.     

Dynamic modelling of Rhodopseudomonas palustris biohydrogen production: Perturbation analysis and photobioreactor upscaling

Developing kinetic models to simulate Rhodopseudomonas palustris biohydrogen production within different configurations of photobioreactors (PBRs) poses a significant challenge. In this study, two types of PBRs: schott bottle-based and vertical tubular-based, were investigated, and three original contributions are presented. Firstly, a mechanistic model was constructed to simulate effects of light intensity, light attenuation and temperature on biomass growth and biohydrogen synthesis, previously not unified for photosynthetic bacteria. Secondly, perturbation analysis was exploited to identify critical parameters influencing the accuracy of the model. Thirdly, two parameters: effective light coefficient and biohydrogen enhancement coefficient, both linked to the PBR's transport phenomena were proposed for process scale-up prediction. By comparing against experimental data, the model's accuracy was confirmed to be high. Moreover, the enhancement of biohydrogen production rate by improved culture mixing and gas removal was also described mechanistically. This provides important advances for future efficient design of PBRs and process online optimisation.     

Electricity and hydrogen co-production from a bio-electrochemical cell with acetate substrate

In the present research, a novel bio-electrochemical cell was designed by using a bipolar membrane to separate the anode and the cathode chambers to overcome the thermodynamic barrier for hydrogen production from acetate. By using this configuration, hydrogen could be produced on cathode without the aid of bias potential or photo irradiation. In the designed bio-electrochemical cell, with 0.1 mol L⁻¹ sulfuric acid as catholyte, acetate could be oxidized on bio-anode with additional electricity generated spontaneously. With an external resistance of 400 Ω, the maximum power density of 0.0145 W m⁻² could be achieved. In the present design, the anode efficiency of 3.9% and cathode efficiency of 41% could be obtained, respectively.     

Optimization of photosynthetic hydrogen production from acetate by Rhodobacter sphaeroides RV

In this study, hydrogen production by Rhodobacter sphaeroides RV from acetate was investigated. Ammonium sulphate and sodium glutamate were used to study the effects of nitrogen sources on photosynthetic hydrogen production. The results showed the optimal concentrations for ammonium sulphate and sodium glutamate were in the range of 0.4–0.8 g/L. Orthogonal array design was applied to optimize the hydrogen-producing conditions of the concentrations of yeast, FeSO₄ and NiCl₂. The theoretical optimal condition for hydrogen production was as follow: yeast 0.1 g/L, FeSO₄ 100 mg/L and NiCl₂ 20 mg/L.     

Photoproduction of hydrogen: Potential dependence of the quantum efficiency as a function of wavelength

The quantum efficiencies (Q) of the photoelectrochemical oxygen evolution reaction on reduced TiO₂ in 0.1 N NaOH have been measured as a function of electrode potential and photon energy (E) over the range 3.2–4.0 eV. Maxima in the Q-E relations over the potential range ?0.4–+1.25 V NHE were observed and the maxima shifted towards lower photon energies as the potential became more positive. This trend can be interpreted in terms of the variation of the absorption coefficient with frequency.     

Heat-acclimatised strains of Rhodopseudomonas palustris reveal higher temperature optima with concomitantly enhanced biohydrogen production rates

Temperature is a critical parameter for bioprocess performance, requiring careful optimisation for peak efficiency. Green biohydrogen production via photofermentation by purple nonsulfur bacteria including Rhodopseudomonas palustris has been extensively researched, yet realisation is limited by comparatively low productivity. We thus assessed the growth and hydrogen productivity of two closely-related strains of R. palustris acclimated to higher temperatures, revealing markedly increased strain-dependent optima than the 30 °C previously accepted. Strain CGA009 grew 53% faster at 35 °C, with 2.4-fold higher hydrogen production rate, while at 40 °C strain ATH 2.1.37 displayed 86% faster growth and 4-fold higher production rate, along with improved specific production and substrate conversion efficiency. These results reaffirm the necessity of pre-acclimation when verifying temperature optima and expand the feasible temperature range for advancement of high-rate biohydrogen production. Further, the superior heat resistance and production capability of strain ATH 2.1.37 raises the potential for further efficiency gains from thermotolerant environmental isolates.     

Electricity generation from acetate and glucose by sedimentary bacterium attached to electrode in microbial-anode fuel cells

Microbial-anode fuel cells (MAFCs) with high electron recovery (>50%) from acetate and glucose have been constructed in this study. By inoculating fresh sedimentary microorganisms into anaerobic anode compartments, a stable current (∼0.42 mA for acetate-fed MAFCs; ∼0.35 mA for glucose-fed MAFCs) is generated from the oxidation of the added organic matter until its concentration decreases to a low level. SEM micrographs indicate that thick biofilms of microbial communities (coccoid cells with a diameter of ∼0.5 μm in acetate-fed MAFCs; rod-shaped cells with a length of 2.0–4.0 μm and a width of 0.5–0.7 μm in glucose-fed MAFCs) completely cover the anode electrodes. These anodophillic biofilms are thought to be responsible for the current generation, and make these microbial-anode fuel cells exhibit good performance even when the growth medium is replaced by a salt buffer without any growth factor. In comparison with those microbial fuel cells that require the addition of artificial electron transfer-mediating compounds, the findings in this study imply a potential way to develop excellent mediator-less MAFCs for electricity generation from organic matter by using substrate-induced anodophillic microbial species.     

Phototrophic hydrogen production from acetate and butyrate in wastewater

Phototrophic hydrogen production was conducted using individual substrates, acetate and butyrate, which are the main products of dark fermentation. Effects of initial pH (ranging 5.0–10.0) and individual substrate concentrations (acetate ranging from 800 to 4100 mg/l, and butyrate ranging from 1000 to 5100 mg/l) on phototrophic hydrogen production were evaluated. The maximum hydrogen yields were 2.5 mol-H₂/mol-acetate at an initial pH of 8.0 treating 800 mg/l of acetate, 3.7 mol-H₂/mol-butyrate at an initial pH of 9.0 treating 1000 mg/l of butyrate. Analyses of DGGE (denaturing gradient gel electrophoresis) profiles of 16S rDNA fragments and FISH (fluorescent in situ hybridization) images show that both phototrophic hydrogen-producing sludges comprised only one predominant species resembling Rhodobacter capsulatus with over 80% relative abundance.     

Enhanced degradation of azo dyes wastewater by S-scheme heterojunctions photocatalyst g-C3N4/MoS2 intimately coupled Rhodopseudomonas palustris with chitosan modified polyurethane sponge carrier

With the development of azo dyes in every aspect of life, azo dyes have brought serious harm to human beings and ecological environment. In this study, a new intimately coupling photocatalysis and biodegradation (ICPB) system was reported: The rod-like graphite carbon nitride-molybdenum disulfide (RCM) of S-scheme was prepared by hydrothermal method and loaded onto the exterior surface of chitosan modified polyurethane sponge (CPU), and Rhodopseudomonas palustris (R. palustris) with metabolic versatility, significant survivability and decomposition to toxic and nonbiodegradable organics was combined inside the carrier. The rod-like RCM and egg-like R. palustris in the CPU were observed, the 13.1° and 27.6° characteristic peaks of g-C₃N₄ and 33.4° and 57.6° characteristic peaks of MoS₂ were detected, N–H, NC, C–S–Mo, N–Mo bonds were confirmed, and E_g = 2.42 eV were calculated in RCM, which confirmed the successful preparation of R. palustris/RCM@CPU. The doping ratio of MoS₂ was 6%, RCM dosage was 0.2 g and chitosan doping ratio was 0.5%, R. palustris/RCM@CPU system showed high degradation efficiency. The removal rates of Congo red, methyl orange and carmine were 99.5%, 97.5% and 99.5%, respectively. The significant removal of R. palustris/RCM@CPU system on azo dyes was due to that the RCM of S-scheme produced strong oxidizing superoxide radical (^.O₂⁻), hydroxyl radical (^.OH) and hole (h⁺), and could oxidize azo groups and benzene rings of azo dyes to form alkane compounds, which were mineralized by R. palustris. The close cooperation among CPU adsorption, RCM oxidation and R. palustris mineralization effectively enhanced the degradation efficiency of R. palustris/RCM@CPU system for a variety of azo dyes. This study would propose a new means for the degradation efficiency of azo dye wastewater.     

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H₂ generation was utilized to study the production of H₂ from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H₂ production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H₂ from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.     

Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond

A new hydrogen-producing photosynthetic bacterium, designated as Rhodovulum sulfidophilum P5, was enriched and isolated from the sludge of a marine shrimp cultivation farm. During fermentation, hydrogen was mainly produced in the late exponential and stationary phases. The optimum culture conditions of strain P5 for hydrogen production were NaCl concentration of 20 g/L, initial pH of 8, temperature of 30 °C, and light intensity of 100 μmol photons/m² s. The maximum hydrogen yield and rate were 2.56 ± 0.18 mol/mol acetate and 19.4 ± 1.6 mL/L h, respectively. Under optimum culture conditions, the hydrogen conversion efficiencies of P5 from acetate, propionate, and butyrate were (64.62 ± 5.05)%, (17.95 ± 0.72)%, and (41.83 ± 2.68)%, respectively. Taken together, these results suggest that this strain has a high salt tolerance and the potential to be used for biohydrogen production and biological treatment of marine organic wastewater.     

Long-term stable hydrogen production from acetate using immobilized Rhodobacter capsulatus in a panel photobioreactor

Biological hydrogen production is attractive since renewable resources are utilized for hydrogen production. In this study, a novel panel photobioreactor (1.4 L) was constructed from Plexiglas with a network of nylon fabric support for agar immobilized bacteria complex. Two strains of Rhodobacter capsulatus DSM 1710 wild-type strain and Rhodobacter capsulatus YO3 (hup⁻, uptake hydrogenase deleted mutant) with cell concentrations of 2.5 and 5.0 mg dcw/mL agar, respectively were entrapped by 4% (w/v) of agar. The system was operated for 72–82 days in a sequential batch mode utilizing acetate as substrate at 30 °C under continuous illumination. Immobilization increased the stability of the photobioreactors by reducing the fluctuations in pH. The pH remained between 6.7 and 8.0 during the process. Both hydrogen yield and productivity were higher in immobilized photobioreactors compared to suspended culture. The highest hydrogen productivities of 0.75 mmol H₂/L/h and 1.3 mmol H₂/L/h were obtained by R. capsulatus DSM1710 and R. capsulatus YO3 respectively.     

Manipulating the hydrogen production from acetate in a microbial electrolysis cell-microbial fuel cell-coupled system

A biological hydrogen-producing system is configured through coupling an electricity-assisting microbial fuel cell (MFC) with a hydrogen-producing microbial electrolysis cell (MEC). The advantage of this biocatalyzed system is the in-situ utilization of the electric energy generated by an MFC for hydrogen production in an MEC without external power supply. In this study, it is demonstrated that the hydrogen production in such an MEC-MFC-coupled system can be manipulated through adjusting the power input on the MEC. The power input of the MEC is regulated by applying different loading resistors connected into the circuit in series. When the loading resistance changes from 10 Ω to 10 kΩ, the circuit current and volumetric hydrogen production rate varies in a range of 78 ± 12 to 9 ± 0 mA m⁻² and 2.9 ± 0.2 to 0.2 ± 0.0 mL L⁻¹ d⁻¹, respectively. The hydrogen recovery (RH₂), Coulombic efficiency (CE), and hydrogen yield (YH₂) decrease with the increase in loading resistance. Thereafter, in order to add power supply for hydrogen production in the MEC, additional one or two MFCs are introduced into this coupled system. When the MFCs are connected in series, the hydrogen production is significantly enhanced. In comparison, the parallel connection slightly reduces the hydrogen production. Connecting several MFCs in series is able to effectively increase power supply for hydrogen production, and has a potential to be used as a strategy to enhance hydrogen production in the MEC-MFC-coupled system from wastes.