Bubble behavior and photo-hydrogen production performance of photosynthetic bacteria in microchannel photobioreactor

A transparent microchannel photobioreactor was manufactured to visualize the colony formation of photosynthetic bacteria (PSB), Rhodopseudomonas palustris CQK 01, as well as the biogas bubble behavior within the microstructure. The results showed that the formation of PSB colony in the interior of microchannels can be divided into four stages: bacteria absorption, bacteria reproduction, morphological transformation and colony formation. It was founded that the microchannel vents immobilized by PSB colony was the favorable sites for the emergence of biogas bubbles. In this work, the effects of substrate concentration and flow rate of the influent solution as well as illumination wavelength and intensity on the photo-hydrogen production performance of the bioreactor were also investigated. The microchannel photobioreactor exhibited a maximal hydrogen production rate of 1.48 mmol/g cell dry weight/h, maximal hydrogen yield of 0.91 mol H₂/mol glucose in all tests at an optimal inlet medium flow rate of 2.8 ml/h and substrate concentration of 50 mmol/l. In addition, photobioreactor showed a highest performance of hydrogen production and substrate consumption at 590 nm illumination wavelength and 5000 lx illumination intensity.     

High-performance biofilm photobioreactor based on a GeO2–SiO2–chitosan-medium-coated hollow optical fiber

To improve hydrogen production performance, this paper describes a novel approach for fabricating a biofilm photobioreactor by adsorption of the photosynthetic bacteria (PSB) Rhodopseudomonas palustris CQK 01 on a hollow optical fiber (HOF) with a GeO₂–SiO₂–chitosan medium (GSCM) coating. The composition of the coating is analyzed using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The biocompatibility of the GSCM-coated HOF and the PSB in the hungry condition are examined. We also quantitatively investigate the biofilm dry weight; protein, polysaccharide, bacteriochlorophyll, carotenoid, and ATP contents of the biofilm cell; and average H₂ production rates. The GSCM-coated HOF exhibits enhanced the biofilm biomass, improved the biofilm activity, and an increased H₂ production rate. The proposed photobioreactor yielded fairly stable long-term performance with a hydrogen production rate of 2.65 mmol/L/h, which is 1.56 and 1.51 times higher than those of photobioreactors with an uncoated HOF and with a fiber having a roughened surface obtained by wrapping it in wire mesh, respectively.     

Improvement of hydrogen production with Rhodopseudomonas palustris CQK-01 by Ar gas sparging

For improving photo-biohydrogen production, a novel gas bubble column photobioreactor with Ar gas sparging was developed for biohydrogen production by purple non-sulfur phototrophic bacteria, Rhodoseudomonas palustris CQK-01. The dissolved hydrogen concentration was in-situ measured by a hydrogen microsensor. Experimental results demonstrated that Ar gas sparging dramatically decreased the dissolved hydrogen concentration, resulting in an improvement in the photo-biohydrogen production performance. Furthermore, effects of the gas flow rate and the time interval of gas sparging were investigated. The results showed that with an increase in the gas flow rate, the hydrogen production performance increased initially due to the reduced dissolved hydrogen concentration and enhanced mass transport, and then it decreased as a result of an increased shear stress. Meanwhile, the short sparging time interval resulted in a low accumulation of dissolved hydrogen in the bioreactor, hence high hydrogen production performance. The optimal hydrogen production rate (5.86 mmol/l/h) and hydrogen yield (3.38 mol H₂/mol glucose) were obtained at the gas flow rate of 10 ml/min, respectively.     

A feasibility study on unsaturated flow bioreactor using optical fiber illumination for photo-hydrogen production

An unsaturated flow bioreactor with Rhodopseudomonas palustris CQK 01 was developed for photo-hydrogen production. The bioreactor developed in this work consisted of a reaction bed packed with spherical glass beads and internal optical fiber that provided uniform light distribution and achieved high light transmission efficiency. Unsaturated conditions were obtained by filling both argon and nutrient solution into the bioreactor. Experimental results demonstrated the feasibility of the biofilm formation in an unsaturated environment. With the formed biofilm, parametric studies on the photo-hydrogen production performance under different liquid flow rates and initial substrate concentrations were conducted. It was found that the performance of the bioreactor came to the optimal state at the liquid flow rate of 500 mL/h. Moreover, such a design enabled the bioreactor to be operated at relatively high glucose concentration without the substrate inhibition, yielding a good biohydrogen production performance.     

Bioflocculation of photo-fermentative bacteria induced by calcium ion for enhancing hydrogen production

The applications of photo-fermentative bacteria (PFB) for continuous hydrogen production are generally subjected to a serious biomass washout from photobioreactor, resulting from poor flocculation of PFB. In this study, through reducing the absolute ζ-potentials of PFB, Ca²⁺ greatly decreased total interaction energy barrier of PFB based on DLVO theory, thus promoted the bioflocculation of Rhodopseudomonas faecalis RLD-53. Average floc size of PFB increased with the Ca²⁺ concentration, and reached maximum 30.07 μm at 4 mmol/l. Consequently, biomass retention capacity of photobioreactor significantly enhanced after 30 min settling with half working volume discharge. In the continuous photo-fermentative sequencing batch reactor, compared with the free cell culture, bioflocculation reached a higher steady-state hydrogen production rate of 879 ml H₂/l/d and hydrogen yield of 2.64 mol H₂/mol acetate, respectively. Therefore, bioflocculation promoted by calcium ion was an effective strategy for retaining PFB in photobioreactor to produce hydrogen continuously.     

Photo-hydrogen production rate of a PVA-boric acid gel granule containing immobilized photosynthetic bacteria cells

The polyvinyl alcohol (PVA)-boric acid gel granule facilitates the light penetration and mass transport as it has the features of the transparency and adequate porous structure. In this work, a hydrogen production bioreactor with the indigenous photosynthetic bacteria (PSB) Rhodopseudomonas palustris CQK 01 immobilized in a PVA-boric acid gel granule is developed to enhance the rate of photo-hydrogen production. Particular attention is paid to exploring the effects of illumination wavelength and intensity, as well as the effects of concentration, flow rate, pH, and temperature of influent substrate solution on the hydrogen production rate. The immobilized PSB gel granule exhibited the maximum hydrogen production rate of 3.6 mmol/g cell dry weight/h in all tests. The experimental results show that the hydrogen production rate of an immobilized PSB granule illuminated at 590 nm is distinctly higher than that at 470 and 630 nm. Photo-inhibition of the gel granule occurs as the long-wavelength illumination intensity exceeds 7000 lux. In addition, there exists an optimal pH of 7.0 and temperature of 30 °C for PSB immobilized in the granule to produce hydrogen. More importantly, the feasibility of PSB immobilized in the PVA-boric acid gel granule for the enhancement of the photo-hydrogen production is demonstrated.     

Enhancing continuous hydrogen production by photosynthetic bacterial biofilm formation within an alveolar panel photobioreactor

Photofermentative hydrogen production at higher rate is desirable to make the technology of biological hydrogen production in practical application. An easy fabricating alveolar panel photobioreactor with high surface-to-volume ratio was proposed in this study to realize biofilm formation and used for developing a continuous bioprocess of hydrogen production. Effects of key operating parameters, i.e. variation in intensity of incident light, initial concentration of carbon substrate and flow rate on the rate of nitrogenase-based H₂ production were investigated using response surface methodology (RSM) with Box-Behnken design. Surface and contour plots of the fitted regression model revealed that optimum H₂ production rate of 57.6 mL/h/L was obtained at 125.9 μE/m²/s incident light intensity at 590 nm light wavelength, 52.4 mM initial concentration of carbon substrate and 209 mL/h flow rate. Regular groove surfaces within this photobioreactor were considered to have mutual effects on enhancement of continuous hydrogen production by enriching bacterial cell density, enhancing mass transfer of carbon substrate to facilitate release of protons and electrons, enhancing removal of molecular H₂, and uniformly distribution of incident light within the photobioreactor for sufficient conversion into ATPs.     

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.     

The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-53 and its application for enhancing continuous hydrogen production

In this work, the kinetic characterization of hydrogen production by the photofermentative bacteria Rhodopseudomonas faecalis RLD-53 was investigated at different growth phase. During entire fermentation, 89.30% of total biomass was accumulated in exponential growth phase, while hydrogen yield was only 1.82 mol H₂/mol acetate at the expense of 51.25% substrate. In the stationary phase, biomass synthesis was minimal (7.51%), and 38.17% of the substrate was directly converted into hydrogen. As a result, hydrogen (59.19%) was mainly produced in stationary phase with highest hydrogen yield of 3.67 mol H₂/mol acetate. Consequently, bacteria in stationary phase were most effective for hydrogen production. Based on these findings, a novel membrane photobioreactor was developed to retain bacteria during stationary phase in reactor through membrane separation. Maximum rate (32.82 ml/l/h) and yield (3.27 mol H₂/mol acetate) of hydrogen production were achieved using membrane photobioreactor under the continuous operation. Therefore, using bacteria in stationary phase as hydrogen producer can offer considerable benefits for enhancing photo-hydrogen production.     

Performance of a groove-type photobioreactor for hydrogen production by immobilized photosynthetic bacteria

The biofilm technique has been proved to be an effective cell immobilization method for wastewater biodegradation but it has had restricted use in the field of photobiological H₂ production. In the present study, a groove-type photobioreactor was developed and it was shown that a groove structure with large specific surface area was beneficial to cell immobilization and biofilm formation of the photosynthetic bacteria on photobioreactor surface as well as light penetration. A series of experiments was carried out on continuous hydrogen production in the groove-type photobioreactor illuminated by monochromatic LED lights and the performance was investigated. The effects of light wavelength, light intensity, inlet glucose concentration, flow rate and initial substrate pH were studied and the results were compared with those obtained in a flat panel photobioreactor. The experimental results show that the optimum operational conditions for hydrogen production in the groove-type photobioreactor were: inlet glucose concentration 10 g/L, flow rate 60 mL/h, light intensity 6.75 W/m², light wavelength 590 nm and initial substrate pH 7.0. The maximum hydrogen production rate, H₂ yield and light conversion efficiency in the groove-type photobioreactor were 3.816 mmol/m²/h, 0.75 molH₂/molglucose and 3.8%, respectively, which were about 75% higher than those in the flat panel photobioreactor.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

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

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

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.     

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

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

Rheological properties of hydrogen fermented food waste

Although rheological properties are essential in scaling up fermenters, there has been no report on hydrogen fermentation of organic solid wastes. In the present work, hydrogen fermentation of food waste (FW) was conducted at various substrate concentrations (10–50 g Carbo. COD/L) and operational pHs (4.5–6.5), and rheological properties were attained. High hydrogen production performance showing over 1.7 mol H₂/mol hexose_added was achieved at 10–40 g Carbo. COD/L and pH 5.5 and 6.0. The viscosity tended to increase with substrate concentration increase, and fermentation led to a significant reduction of viscosity. Before fermentation, zero viscosity and infinite viscosity of FW ranged 6.8–1274.1 mPa s and 2.2–58.1 mPa s, which were reduced to 10.4–346.2 mPa s and 1.1–5.3 mPa s after fermentation, respectively. Compared to substrate concentration, operational pH showed a less effect on rheological properties. At the similar volatile solids concentration range, hydrogen-fermented FW showed lower values of rheological properties than that of anaerobic digester sludge. Based on the agitation speed values assuring turbulent condition (complete mixing), the energy required for agitation can be reduced by 30–67% by lowering the applied agitation speed with fermentation time.     

Hydrogen production using Rhodobacter sphaeroides (O.U. 001) in a flat panel rocking photobioreactor

Photofermentative hydrogen production is challenged by the photobioreactor design that can overcome poor light penetration, agitation and temperature control. Flat panel reactors have been reported to have several advantages over other reactors. But they are limited to a suitable type of agitation system when using it for hydrogen production. The aim of the present study is to develop and improve a flat panel reactor that can overcome the problem of agitation with a rocking motion. Studies with Rhodobacter sphaeroides O.U. 001 resulted in a cumulative hydrogen production of 492 ± 10 mL with maximum production rate of 11 mL L⁻¹ h⁻¹, substrate (malic acid) conversion efficiency of 44.4% and light conversion efficiency of 3.31%. The mixing time of the reactor was found to be around 17 s with a power input of 100-275 W/m³. Though the entire reactor was in motion the energy spent for the rocking motion was found to be quite low.     

Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort

A newly enriched marine phototrophic bacterial consort was studied for its capability of hydrogen production in batch cultivations using butyrate as the sole carbon source. Analyses of denaturing gradient gel electrophoresis (DGGE) profiles showed that the mixed bacterial consort consisted mainly of Ectothiorhodospira, Sporolactobacillus, and Rhodovulum. Important parameters investigated include temperature, light intensity, initial pH, and butyrate concentration. The pH of the culture medium significantly increased as fermentation proceeded. Optimal cell growth was observed at temperature of 25–35 °C, light intensity of 80–120 μmol photons/m² s, initial pH of 8, butyrate concentration of 20–40 mmol/l. Optimal conditions for hydrogen production were 30 °C, light intensity of 80 μmol photons/m² s, initial pH 8. The increase of butyrate concentration (10–50 mmol/l) resulted in higher hydrogen production, but the yield of hydrogen production (mol H₂/mol butyrate) gradually decreased with increasing butyrate concentration. The maximal hydrogen yield and hydrogen production rate were estimated to be 2.52 ± 0.12 mol H₂/mol butyrate and 19.40 ± 2.32 ml/l h, respectively. These results indicate that optimization of the culture conditions resulted in a simultaneous increase in biohydrogen production and cell growth.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

Biohydrogen production by Rhodobacter capsulatus Hup mutant in pilot solar tubular photobioreactor

In this study, a pilot solar tubular photobioreactor was successfully implemented for fed batch operation in outdoor conditions for photofermentative hydrogen production with Rhodobacter capsulatus (Hup⁻) mutant. The bacteria had a rapid growth with a specific growth rate of 0.052 h⁻¹ in the batch exponential phase and cell dry weight remained in the range of 1–1.5 g/L throughout the fed batch operation. The feeding strategy was to keep acetic acid concentration in the photobioreactor at the range of 20 mM by adjusting feed acetate concentration. The maximum molar productivity obtained was 0.40 mol H₂/(m³ h) and the yield obtained was 0.35 mol H₂ per mole of acetic acid fed. Evolved gas contained 95–99% hydrogen and the rest was carbon dioxide by volume.     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.