The effect of cycA overexpression on hydrogen production performance of Rhodobacter sphaeroides HY01

The gene cycA encodes a periplasmic protein, cytochrome c₂ (cyt c₂), which dominates electron transfer from the membrane-bound ubiquinol: cyt c₂ oxidoreductase (cyt bc₁) to the photosynthetic reaction center, contributing to the production of transmembrane proton potential and then the synthesis of ATP. For photosynthetic bacteria, the total energy supply for light anaerobic growth and hydrogen production comes from photophosphorylation. As a result, the key protein encoding gene plays an important role in hydrogen production. To figure out the specific effect of cycA expression level on H₂ production ability of Rhodobacter sphaeroides HY01, cycA-expression plasmids derived from pRK415 and pBBR1MCS-2 were constructed and then crossed into the parent strain R. sphaeroides HY01 for H₂ production test. And further verification by RT-PCR suggested that there was about 20% enhancement of cycA expression level by pBBR1MCS-2 where the H₂ production performance of corresponding strain was improved by 6–8% compared with blank control. In contrast, cycA expression level was about 3.4 folds by vector pRK415 compared with control strain, but corresponding strains showed slightly depressed H₂ production performance. Besides, the mutant XJ01 with cycA gene overexpressing by 70% in the genome of HY01(hupSL:cycA) also showed positive effect on hydrogen production performance. The results demonstrated that slightly overexpression of cycA could enhance the hydrogen production rate, but too much higher level of cycA-expression could show negative effect on H₂ production performance of R. sphaeroides HY01.     

The exploration of hydrogen production promoting mechanism of Rhodobacter sphaeroides by expressing tetA under tetracycline stress

Photo-fermentative hydrogen production rate improving has been a key limiting link in the field of biological hydrogen production by photosynthetic bacterium (PSB) in recent years. In our previous study, apparent hydrogen production enhancement was observed in R. sphaeroides HY01 with tetA expressed under tetracycline (Tc), which attracted our interests. It was found that Tc-resistant strains always presented stable hydrogen production promotion on the maximum hydrogen production rate (R_m), which was approximately 30% with Tc addition. Figuring out the underlying connections between Tc resistance and hydrogen production of R. sphaeroides may provide new methods for obtaining engineered PSB with high hydrogen production performance. In this study, basing on experimental tests, hydrogen promotion of resistant strain was found to be Tc dose-dependent. Some relevant gene expression differences on hydrogen production of Rodobacter sphaeroides induced by the expression of TetA and the addition of Tc were also tested and discussed.     

Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum

The genes coding for two PII-like proteins, GlnB and GlnK, which play key roles in repressing the nitrogenase expression in the presence of ammonium ion, were interrupted from the chromosome of Rhodobacter sphaeroides. The glnB–glnK mutant exhibits the less ammonium ion-mediated repression for nitrogenase compared with its parental strain, which results in more H₂ accumulation by the mutant under the conditions. Rhodospirillum rubrum produces H₂ by both nitrogenase and hydrogenase. R. rubrum containing the recombinant pRK415 with an insert of hydC coding for its own Fe-only hydrogenase showed twofold higher accumulation of H₂ in the presence of pyruvate under photoheterotrophic conditions, which was not observed in the absence of pyruvate. The same was true with R. rubrum containing the recombinant pRK415 cloned with hydA coding for Fe-only hydrogenase of Clostridium acetobutylicum. Thus, Fe-only hydrogenase requires pyruvate as an electron donor for the production of H₂.     

Enhancement on hydrogen production performance of Rhodobacter sphaeroides HY01 by overexpressing fdxN

Ferredoxin I (FdI), encoded by fdxN gene, is proved to be the main electron donor of nitrogenase for hydrogen production. In this work, fdxN gene overexpression was implemented in a mutant MHY01, which was constructed by inserting fdxN gene into the hupSL region in Rhodobacter sphaeroides HY01 genome. Its photo-fermentative H₂ production performance was studied. The results showed that the expression level of fdxN and nitrogenase activity in MHY01 (hupSL::fdxN) were enhanced by 177% and 61.7% respectively compared with that of wild type HY01. Using 25 mM acetate and 34 mM butyrate as carbon source and 6 mM l-glutamate as nitrogen source, the maximum H₂ production rate was 156.1 mL/(L·h), which was increased by 50.7% compared with that of HY01. The maximum H₂ production rates of MHY01 were enhanced by 30.0%, 52.5% and 50.7% compared with those obtained from HY01 at the inoculation size of 5%, 10% and 15% respectively. The results suggested that overexpressing fdxN could enhance the nitrogenase activity and H₂ production performance of purple non-sulfur bacteria. The abundancy of ferredoxin I might limit the efficiency of electron transfer flux associated with the biohydrogen production process.     

Single-stage photo-fermentative hydrogen production from hydrolyzed straw biomass using Rhodobacter sphaeroides

The hydrogen production utilizing photosynthetic and anaerobic bacteria in two-stage approach has many drawbacks, such as shortage of raw materials and complexity of operations. Accordingly, we aimed to develop a simple one-stage H₂ production protocol using the depolymerization of maize straw cellulose as a cheap carbon source. R. sphaeroides HY01 and its mutant (Hup^?) were studied regarding their H₂ production under different culture conditions. Further study using two model sugars, their combination, and straw hydrolysate as carbon sources was conducted to determine the effects of substrate on H₂ production. When using the straw hydrolysate as carbon source, the pH remained in a range of 7.1–7.6, whereas it dropped to 5.4–7.4 when using the model sugars, and the former biomass value was greater. The H₂ production performance of the mutant was significantly better than that of HY01. One-step photo-fermentative H₂ production was superior when using straw hydrolysate as opposed to the simple model sugars, and its yield was up to 4.62 mol H₂·mol^?1 reducing sugar.     

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.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Evaluating biohydrogen production by Clostridium hydrogenum sp. nov. strain CUEA01 isolated from mangrove sediments in Thailand

The hydrogen (H₂) fermentative Clostridium hydrogenum sp. nov. strain CUEA01 was isolated from a mangrove sediment in Thailand. Genome sequencing and analysis revealed a genome size of 5,501,482 bp that encoded for 3,292 predicted protein coding genes with annotated functional assignments and many genes associated with carbon utilization and H₂ evolution. The H₂ production performance was evaluated in batch fermentation, and revealed that this strain can grow and produce H₂ at a broad range of temperatures (15–40 °C), pH (4–10), and initial glucose concentrations (5–60 g/L). The maximum H₂ yield (3.11 mol_H2/mol_glucose) was obtained at 37 °C, pH 8, and an initial glucose concentration of 10 g/L. Furthermore, this strain could utilize various carbon sources, including xylose, xylan, starch, mannose, glycerol, and avicel cellulose, amongst others. Additionally, CUEA01 was compatible with agro-industrial wastes and could achieve a maximum CHP of 4639 mL/L and 4024 mL/L from sugarcane molasses and cassava pulp, respectively. This demonstrates that CUEA01 has a potential for H₂ fermentation from complex organic wastes since it can secrete enzyme cocktails that consolidate the fermentation process.     

A newly isolated Rhodobacter sphaeroides HY01 with high hydrogen production performance

A newly isolated strain, HY01, was identified to be Rhodobacter sphaeroides by phylogenetic analysis. Some DNA sequence data indicate that it is highly similar to R. sphaeroides ATCC 17029. The effects of initial pH values, nitrogen sources and carbon sources on hydrogen production were studied. The results showed that pH 7.25 is optimum for its hydrogen production. Among the nitrogen sources of glutamic acid, yeast extract, glycine, ethanolamine and l-aspartic acid, the maximum hydrogen production rate of 98.0 ± 6.2 mL/(Lh) using 10 mM glutamic acid and maximum hydrogen yield of 7012.5 ± 150 mL/L using 5 mM glutamic acid was obtained. Maximum hydrogen production rates of 148.7 ± 4.6 mL/(Lh) and 94.8 mL/(Lh) were obtained under 10 klux and 5 klux light intensity using 7 mM glutamic acid as nitrogen source, respectively. Compared with the reported data, it shows high hydrogen production performance and is a good candidate for further study.     

New tolerant strains of purple nonsulfur bacteria for hydrogen production in a two-stage integrated system

In this study we described the isolation of eight new strains of purple non-sulfur bacteria resistant to salinity ≥30 g L⁻¹ and high concentration of VFAs (200 mM). These strains were characterized by their general physiological properties and the occurrence of hupSL genes. Some correlation was observed between the rate of H₂ photoproduction, the absence of hupSL genes and hydrogenase activity. Two fast-growing strains without hupSL genes showed high nitrogenase activity and hydrogen accumulation during growth on Ormerod medium. These strains were capable of H₂ photoproduction using non-treated dark culture (75% in water) after dark fermentation of starch at 30 g L⁻¹, unlike control strains, Rhodobacter capsulatus B10 and Rb. sphaeroides GL. New N7 and 13 strains identified as Rb. sphaeroides can be recommended for application in a two-stage H₂ production system.     

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.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

The distillers grains with solubles as a perspective substrate for obtaining biomass and producing bio-hydrogen by Rhodobacter sphaeroides

The photosynthetic purple non-sulfur bacterium Rhodobacter sphaeroides MDC6521, isolated from Armenian mineral spring, and distillers grains with solubles (DGS) (by-product of bio-ethanol fermentation) were applied for obtaining biomass and producing bio-hydrogen (H₂) upon illumination. During growth on diluted DGS media H₂ production was started at 24 h growth, whereas H₂ photoproduction by R. sphaeroides cells, grown on Ormerod medium, was detected after 48 h. Moreover, the R. sphaeroides produced considerably more H₂ during DGS photo-fermentation: the H₂ yields from 2- fold and 5-fold diluted media were ∼4.6–5.5-folds higher in comparison with control. H₂ yield has decreased at higher dilution. The growth yields of R. sphaeroides cells, grown in the 2–5-folds diluted DGS media, were considerably higher than those of control cells, grown in Ormerod medium. The results can provide with new cheaper and more effective source of biomass and bio-hydrogen and as well as solve the problem of ethanol by-product utilization.     

Growth characteristics and hydrogen production by Rhodobacter sphaeroides using various amino acids as nitrogen sources and their combinations with carbon sources

Some amino acids (alanine, asparagine, glutamate, glycine, proline, and tyrosine) were used as nitrogen sources in combination with carbon sources (succinate and malate) to study growth properties and H₂ production by purple non-sulfur bacterium Rhodobacter sphaeroides strains A-10 and D-3. Both strains produced H₂ in succinate–glutamate and malate–glutamate media. Succinate was a better carbon source than malate. In comparison with strain D-3, strain A-10 was able to utilize proline, alanine or tyrosine as nitrogen sources in succinate medium and to produce H₂. Both strains were unable to produce H₂ in the presence of asparagine or glycine as nitrogen sources. N,N′-dicyclohexylcarbodiimide, the F₀F₁-ATPase inhibitor, led to marked inhibition of H₂ production activity of R. sphaeroides. The results suggest that the R. sphaeroides cells growth can be achieved by the use of a large diversity of substrates but only some of them can increase the H₂ production rate.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

Photocatalytic decomposition of H2S to produce H2 over CdS nanoparticles formed in HY-zeolite pore

The H₂ production rate from H₂S photocatalytic decomposition under visible light irradiation (λ > 400 nm) over CdS nanoparticules formed in HY-zeolite pore (named CdS/HY) was much higher compared to the commercial bulk CdS. The CdS/HY photocatalyst was characterized by UV–Vis, XRD, FT-IR, N₂ adsorption, SEM and HRTEM. The blue shift from bulk which confirmed CdS nanoparticles located in the pore of HY-Zeolite (named HY). Photocatalytic activity and surface area were enhanced by such structures.     

Enhanced bio-hydrogen production from sugarcane juice by immobilized Clostridium butyricum on sugarcane bagasse

Immobilized Clostridium butyricum TISTR 1032 on sugarcane bagasse improved hydrogen production rate (HPR) approximately 1.2 times in comparison to free cells. The optimum conditions for hydrogen production by immobilized C. butyricum were initial pH 6.5 and initial sucrose concentration of 25 g COD/L. The maximum HPR and hydrogen yield (HY) of 3.11 L H₂/L substrate·d and 1.34 mol H₂/mol hexose consumed, respectively, were obtained. Results from repeated batch fermentation indicated that the highest HPR of 3.5 L H₂/L substrate·d and the highest HY of 1.52 mol H₂/mol hexose consumed were obtained at the medium replacement ratio of 75% and 50% respectively. The major soluble metabolites in both batch and repeated batch fermentation were butyric and acetic acids.     

Ultrasonic pretreatment for an enhancement of biohydrogen production from complex food waste

The efficacy of ultrasonication pretreatment method for complex food waste prior to anaerobic digestion is evaluated for enhancement of H₂ yield (HY) and rate (R). The RSM results showed that the ultimate H₂ production increased with increasing TS content and ultrasonication time (UT). Desirability function integrated with RSM predicted an optimum condition of TS and UT as: 8% TS and 12 min, for maximization of HY and R. The highest HY, 149 mL/g VS_added, and R, 5.23 mL/h, were achieved during the verification test at optimized conditions. Furthermore, a significant decreased lag phase followed by highest molar HBu/HAc ratio (2.2) was also achieved at optimized conditions with lowest specific energy input (13,500 kJ/kg TS). The significant relative enhancement of HY, 75%, and R, 104%, implies that ultrasonically pretreated complex food waste with higher TS loading is about 1.7–2.1 times more effective for enhanced bioH₂ production compared to unsonicated food waste.     

Unravelling the enhancement of biohydrogen production via adding magnetite nanoparticles and applying electrical energy input

Magnetite nanoparticles (MNP)-caused enhancements for H₂ yield (HY) are usually justified based on oxidation-reduction potential (ORP), iron ions (Fe^+2/+3) concentration, and enzymatic activity, that is pH-dependent. However, the questions “If pH, ORP, and Fe^+2/+3 impacts are excluded, will MNP-caused HY enhancement be still present? and how electrical energy input (EEI) affects HY?” are still unanswered. Herein, control, MNP-supplemented, EEI-applied, FeCl₃-, and Na₂S-supplemented batches, referred as G1, G2, G3, G5, and G6, respectively were conducted, under fixed pH (6.0 ± 0.1). G5 and G6 targeted quantifying sole impact of Fe^+2/+3, and ORP, respectively on HY. G1, G2, G3, G5, and G6 achieved HY values of 1.10 ± 0.05, 1.66 ± 0.07, 1.38 ± 0.06, 1.18 ± 0.04, and 1.16 ± 0.05 mol H₂/mol_hexose, respectively. Neither Fe^+2/+3 release nor ORP reduction significantly affected HY. Further, Clostridium amylolyticum dominance was almost similar among G2, and G3. Metabolites flux analysis and functional genes’ prediction highlighted that G2 achieved highest hydrogenase expression and lowest homoacetogenic H₂ consumption.     

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.