Photo-fermentative hydrogen production performance of a newly isolated Rubrivivax gelatinosus YP03 strain with acid tolerance

Screening and excavating new photosynthetic bacteria with excellent hydrogen production performance is extremely important for improving the photo-fermentative hydrogen production. A new photosynthetic bacterium YP03 was isolated and identified to be Rubrivivax gelatinosus by morphological characterization and phylogenetic analysis. The effects of several key factors on hydrogen production performance were carried out. The results indicated that YP03 strain showed a preference for the carbon sources, and 5375 ± 398 mL/L of maximum hydrogen yield was obtained using butyrate medium. Meanwhile, YP03 strain could use several nitrogen sources to produce hydrogen, and glutamic acid was the optimum nitrogen source for hydrogen produced. Furthermore, YP03 exhibited better hydrogen production performance at initial pH 7.0, reaction temperature 33 °C and light intensity 5000 lux, and the maximum hydrogen production rate was 108.3 ± 12.4 mL/(Lh), which was relatively high compared with the previous reports by R. gelatinosus. Especially, the proper pH for hydrogen production by YP03 ranged from weak acid to neutral (6.5–7.0) and it still could produce hydrogen at pH 5.5 showing the characteristic of acid tolerance. It suggested that YP03 is a potential candidate for the integration of dark- and photo-fermentative hydrogen production. These findings contribute to our understanding of YP03 strain and provide a prospective photosynthetic bacterium for efficient hydrogen production in future research.     

Photo-fermentative hydrogen production from mixed substrate by mixed bacteria

Photo-fermentative H₂ production by mixed bacteria and pure bacterium Rhodopseudomonas faecalis RLD-53 using single and mixed substrate as carbon source was investigated in batch culture. Experimental results showed that 60 mmol/L acetate was the optimal concentration for mixed bacterial H₂ production and maximum cumulative H₂ volume was 2468 ± 123 mL H₂/L-culture. It was also found that propionate or butyrate was a key factor for enhancing H₂ production in mixed substrate system. Photo-H₂ production can be greatly promoted when proper concentration of propionate and butyrate were added into acetate medium as mixed substrate and a higher H₂ yield of 2931 ± 146 mL H₂/L-culture was obtained. In addition, it was worth noting that when the strain RLD-53 was added into mixed bacteria with different concentration ratios, H₂ yield did not yet increase. Interestingly, H₂ production capacity gradually decreased with ratio of strain RLD-53 to mixed bacteria from 8:0 to 4:4, and then gradually increased from 4:4 to 0:8. This implied that the competition relationship between strain RLD-53 and mixed bacteria in substrate utilization strongly influenced their H₂ production.     

Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H₂ production after pretreatment. However, the pretreatment methods have different influence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/cellusase. The hydrolysis rates increased from 45.51% by ddH₂O-treatment to 60.79% by diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H₂ production vary among different hydrolysates. Photo-fermentative H₂ production of strain R. capsulatus JL1 and mutant JL1601 (cheR2^-) with hydrolysates were investigated. The maximum H₂ yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H₂ yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2^-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2^-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH₂O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H₂ production for mutant JL1601 (cheR2^-) but not for strain JL1.     

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.     

Improved photo-fermentative hydrogen production by biofilm reactor with optimizing carriers and acetate concentration

To enhance photo-fermentative hydrogen production (PFHP), biofilm reactor (BR) was employed as an ideal strategy with optimization on key factors of acetate concentration and carriers in this work. Optimal conditions for hydrogen production were acetate concentration of 4 g/L and carriers (silicon sheet) of 10 cm × 1 cm at amount of 1 piece. Biofilm formed on silicon sheet strongly improved hydrogen production compared with control reactor (CR). Cumulative hydrogen volume was enhanced about 20% from 2850 ± 130 mL/L of CR to 3349 ± 153 mL/L of BR and hydrogen yield was increased 20% from 2.61 ± 0.13 mol H₂/mol acetate of CR to 3.06 ± 0.15 mol H₂/mol acetate of BR at 4 g/L acetate. Protein and deoxyribonucleic acid (DNA) were important components to form the biofilm and they occupied 90% of extracellular polymeric substances (EPS). In particular, DNA, nearly 50% content of EPS, likely indicated a substantial contribution to biofilm formation and bacterial communication. Moreover, it suggested biofilm could regulate free cells to decline EPS secretion for improved hydrogen production. This work indicates BR could be a promising and economic strategy to enhance hydrogen production by photo-fermentation.     

Assessment via the modified gompertz-model reveals new insights concerning the effects of ionic liquids on biohydrogen production

Lignocellulosic biofuel, in particular hydrogen gas production is governed by successful feedstock pretreatment, hydrolysis and fermentation. In these days, remarkable attention is paid to the use of ionic liquids to make the fermentable regions of lignocellulose biomass more accessible to the biocatalysts. Although these compounds have great potential for this purpose, their presence during the consecutive fermentation stage may pose a threat on process stability due to certain toxic effects. This, however, has not been specifically elaborated for dark fermentative biohydrogen generation. Hence, in this work, two common imidazolium-type ionic liquids (1-butyl-3-methylimidazolium acetate, ([bmim][Ac]) and 1-butyl-3-methylimidazolium chloride, ([bmim][Cl])) were employed in mixed culture biohydrogen fermentation to investigate the possible impacts related to their presence and concentrations. The batch assays were evaluated comparatively via the modified Gompertz-model based on the important parameters characterizing the process, namely the biohydrogen production potential, maximum biohydrogen production rate and lag-phase time.     

The prospects of brewery waste application in biohydrogen production by photofermentation of Rhodobacter sphaeroides

Selection of the source for biohydrogen (H₂) production is essential, since it affects bacterial metabolism. Pure organic substrates give fast H₂ generation with high yields, but they increase the production cost. Using industrial wastes as a source provides inexpensive energy generation with simultaneous waste utilization. H₂ production by photofermentation of Rhodobacter sphaeroides is monitored during cultivation on brewery waste. Maximum specific growth rate is observed for 5–10% waste containing media. H₂ production by cells, grown on waste, is detected at 48–96 h of growth; it is comparable or higher than that of control medium with expensive carbon and nitrogen sources. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity of R. sphaeroides membrane vesicles from growth on waste containing media is 1.6–1.7-fold higher compared to control, correlating with enhanced H₂ production. Growth medium containing optimal amount of wastes may be a successful alternative to expensive media for high H₂ yield in R. sphaeroides during photofermentation.     

Biohydrogen production relationship to biomass composition,growth, temperature and nitrogenase isoform in the anaerobic photoheterotrophic diazotroph Rhodopseudomonas palustris

Hydrogen (H₂) gas is an obligatory byproduct of nitrogen (N₂) reduction during biological nitrogen fixation by the metalloenzyme nitrogenase. Despite significant efforts, diazotrophic H₂ production rates remain too low to compete with fossil fuel-derived H₂. Here, we investigate the role of temperature (14, 19, 30 °C), carbon metabolism (acetate or succinate as carbon source), and nitrogenase isoform (molybdenum, vanadium, iron-only nitrogenase) in controlling N₂ reduction and H₂ production rates in the model anaerobic photoheterotroph Rhodopseudomonas palustris.Rates of H₂ production are primarily controlled by growth rate and secondarily by nitrogenase enzymology. The iron-only nitrogenase exhibits the highest H₂:N₂ stoichiometries (6.3–12.7); H₂:N₂ stoichiometries for molybdenum and vanadiumnitrogenases are similarly lower (2.5–4.1 and 2.6–4.3, respectively) and uncorrelated with growth rate or temperature. Hydrogen inhibition of growth is lower than H₂ inhibition of purified nitrogenase. These results help provide a framework for optimizing physical and metabolic conditions in diazotroph-based biohydrogen production efforts.     

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.     

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.     

Enhanced photo-fermentative hydrogen production of Rhodopseudomonas sp. nov. strain A7 by biofilm reactor

To achieve stable and efficient photo-fermentative hydrogen production, this work investigated photo-fermentative hydrogen production by forming biofilm on the surface of carrier in the biofilm reactor (BR). Results showed the hydrogen production performance was greatly improved by formed biofilm. The time of hydrogen production and efficiency of substrate utilization were enhanced obviously compared to the control reactor (CR). When the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 ± 87 mL/L and 1.44 ± 0.07 mol H₂/mol acetate, respectively. However, in the BR hydrogen production volume of 3028 ± 150 mL/L and hydrogen yield of 2.52 ± 0.13 mol H₂/mol acetate were obtained, which were enhanced about 75% compared to that of the CR. The time of hydrogen production extended from 7 days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB).     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures

Dark fermentation using mixed cultures is an attractive biological process for producing hydrogen (H₂) from lignocellulosic biomass at a low cost. Physicochemical pretreatment is generally used to convert lignocellulosic materials into monosaccharides. However, the processes also involved release degradation byproducts which can, in turn, inhibit microbial growth and metabolism and, hence, impact substrate conversion. In this study, the impact on H₂ production of lignocellulose-derived compounds (i.e. furan derivatives, phenolic compounds and lignins) was assessed along with their effect on bacterial communities and metabolisms. Batch tests were carried out using xylose as model substrate (1.67 mol_H2 mol_xylose⁻¹ in the control test). All the putative inhibitory compounds showed a significant negative impact on H₂ production performance (ranging from 0.34 to 1.39 mol_H2 mol_xylose⁻¹). The H₂ yields were impacted more strongly by furan derivatives (0.40–0.51 mol_H2 mol_xylose⁻¹) than by phenolic compounds (1.28–1.39 mol_H2 mol_xylose⁻¹). Except for the batch tests supplemented with lignins, the lag phase was shorter for inhibitors having the highest molecular weight (8 days versus 22 days for the lowest MW). Variability of the lag phase was clearly related to a shift in bacterial community structure, as shown by multivariate ordination statistics. The decrease in H₂ yield was associated with a decrease in the relative abundance of several H₂-producing clostridial species. Interestingly, Clostridium beijerinkii was found to be more resistant to the inhibitors, making this bacterium an ideal candidate for H₂ production from hydrolyzates of lignocellulosic biomass.     

Influence of butyrate on fermentative hydrogen production and microbial community analysis

The effect of butyrate on hydrogen production and the potential mechanism were investigated by adding butyric acid into dark fermentative hydrogen production system at different concentrations at pH range of 5.5–7.0. The results showed that under all the tested pH from 5.5 to 7.0, the addition of butyric acid can inhibit the hydrogen production, and the inhibitory degree (from 10.5% to 100%) increased with the increase of butyric acid concentration and with the decrease of pH values, which suggested that the inhibition effect is highly associated with the concentration of undissociated acids. Substrate utilization rate and VFAs accumulation also decreased with the addition of butyric acid. The microbial community analysis revealed that butyrate addition can decrease the dominant position of hydrogen-producing microorganisms, such as Clostridium, and increase the proportion of other non-hydrogen-producing bacteria, including Pseudomonas, Klebsiella, Acinetobacter, and Bacillus.     

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.     

Bio-hydrogen production by a new isolated strain Rhodopseudomonas sp. WR-17 using main metabolites of three typical dark fermentation type

In this work, a new strain WR-17 was isolated for photo-fermentative hydrogen production and its hydrogen production capacity was investigated by utilizing main liquid byproducts of three dark fermentation types in batch culture. Experimental results indicated that strain WR-17 was identified as genus Rhodopseudomonas and maximum hydrogen yield of 2.42 mol H₂/mol acetate was obtained when the acetate was used as sole carbon source. Strain WR-17 had an excellent ability of using mixed short chain acids of three typical fermentations such as acetate and ethanol, acetate and butyrate, acetate and propionate. Result demonstrated that the metabolites of butyric acid-type fermentation as substrate is fitting to produce hydrogen and maximum cumulative hydrogen volume of 2156 ml/L-medium was obtained when acetate of 30 mmol/L and butyrate of 15 mmol/L were used. Therefore, butyric acid-type fermentation has great potential for further obtaining high hydrogen yield by the combining photo-fermentation.     

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H₂) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H₂ yield (HY) of 0.65 mol H₂/mol acetate- and 2.50 mol H₂/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H₂/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H₂ production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H₂ production. When pH was controlled to 7.0, strain KD131 wt evolved more H₂ by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H₂ production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH₄)₂SO₄. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H₂ production in expending reducing power.     

Impact of 5-hydroxy methyl furfural on continuous hydrogen production from galactose and glucose feedstock with periodic recovery

A continuous stirred tank reactor (CSTR) was operated for more than 120 days with fixed hydraulic retention time of 6 h at mesophilic temperature along with a periodic recovery phase towards hydrogen production and stimulated by the existence of 5-hydroxy methyl furfural concentration (5-HMF). Interestingly, CSTR mixed with a small amount of 5-HMF, range of 0.3–0.6 g/L showed at least 50% higher hydrogen production rate than control without 5-HMF. However, when 5-HMF concentration was higher than 0.6 g/L, the performance was significantly inhibited. The bacterial community shifted by 5-HMF from Clostridium-dominated to Lactobacillus-dominated population. Regardless of the remain 5-HMF concentration in CSTR, the microbial community and hydrogen producing performance were restored by stop mixing the 5-HMF from the feedstock. The high-rate hydrogen production of 20.0 ± 1.8 L H₂/L/d was achieved in the presence of 5-HMF using the threshold information and recovery strategy.     

Steam reforming of propionic acid: Thermodynamic analysis of a model compound for hydrogen production from bio-oil

The thermodynamic equilibrium of steam reforming of propionic acid (HPAc) as a bio-oil model compound was studied over a wide range of reaction conditions (T = 500–900 °C, P = 1–10 bar and H₂O/HPAc = 0–4 mol/mol) using non-stoichiometric equilibrium models. The effect of operating conditions on equilibrium conversion, product composition and coke formation was studied. The equilibrium calculations indicate nearly complete conversion of propionic acid under these conditions. Additionally, carbon and methane formation are unfavorable at high temperatures and high steam to carbon (S/C) ratios. The hydrogen yield versus S/C ratio passes a maximum, the value and position of which depends on temperature. The thermodynamic equilibrium results for HPAc fit favorably with experimental data for real bio-oil steam reforming under same reaction conditions.     

Regulation of the NADH supply by nuoE deletion and pncB overexpression to enhance hydrogen production in Enterobacter aerogenes

In this study, seven mutants from E. aerogenes IAM1183 wildtype were constructed via different strategies including deletion of lactate dehydrogenase, disruption of NADH dehydrogenase gene nuoE, overexpression of pncB and a combination of both to regulate of the NADH supply to enhance hydrogen production. Compared with the parental strain, the hydrogen yields of the strains IAM1183-E, IAM1183-L and IAM1183-EL increased by 23.3, 81.7 and 97.9%, respectively. When the pncB gene was overexpressed, the hydrogen yield of IAM1183/P, IAM1183-E/P, IAM1183-L/P and IAM1183-EL/P increased by 39.0, 6.5, 5.9, and 5.1% compared with the respective original knockout strains. Among them, the total hydrogen yield of strain IAM1183-EL/P with highest production efficiency was 58% higher than IAM1183. Further metabolite analysis indicated that the knockout of nuoE and ldhA, combined with the overexpression of pncB, resulted in a redistribution of the metabolic fluxes in E. aerogenes, which led to an improvement of the hydrogen yield.