Yeast extract as an effective nitrogen source stimulating cell growth and enhancing hydrogen photoproduction by Rhodobacter sphaeroides strains from mineral springs

The suitability and limitation of yeast extract as nitrogen source to support cell growth and to enhance hydrogen photoproduction by Rhodobacter sphaeroides strains MDC6521 and MDC6522 isolated from mineral springs in Armenia was investigated during the anaerobic growth. Yeast extract (2 g L⁻¹) was indicated to be an effective nitrogen source for bacterial cell growth stimulation and enhanced H₂ production (compared to glutamate). Both strains followed similar growth patterns in medium with yeast extract as nitrogen source and succinate or malate as carbon source. The highest growth rate was obtained for bacterial cells with yeast extract: the latter added gave a stimulated (2–3.5 fold) growth rate than using glutamate. R. sphaeroides suspension oxidation–reduction potential (ORP), which was measured with a platinum electrode, decreased down to low negative values with nitrogen source for both strains. ORP decreased down to more negative values (−610 ± 25 mV) in the presence of yeast extract than when adding glutamate (−405 ± 15 mV) compared to the control (without nitrogen source addition): the significant decrease of ORP indicated enhanced (∼6 fold) H₂ yield. The noticeable ORP decrease measured with the titanium-silicate electrode and simultaneously the increase of extracellular pH ([pH]_out) were observed; ORP was more negative at alkaline [pH]_out. Thus, the optimal culture conditions with nitrogen and carbon sources for bacterial growth stimulation and enhanced H₂ production were established. The ORP decrease together with the increase of [pH]_out point out a significant role of reduction processes in cell growth and ability of bacteria to live.     

Optimization of photo-hydrogen production based on cheese whey spent medium

Cheese whey wastewater diluted to 10 g lactose/L was initially subjected to dark-fermentation by Enterobacter aerogenes MTCC 2822, and the VFAs-rich spent medium (acetic acid 1900 mg/L, butyric acid 537 mg/L, and traces of propionic acid) was subjected to photo-fermentation through enrichment by Ni²⁺ (0–8 μmol/L), Fe²⁺ (0–100 μmol/L) or Mg²⁺ (0–15 mmol/L) in batch mode by Rhodopseudomonas BHU 01 strain. The maximum cumulative H₂ production (144 ml) and yield (58 mmol) was obtained at 4 μmol Ni²⁺/L. Likewise, Fe²⁺ (60 μmol/L) resulted in maximum cumulative H₂ production (139 ml) and yield (56 mmol). Nevertheless, 6 mmol of Mg²⁺ did not significantly affect H₂ production (110 ml) or yield (44 mmol); the latter value in close proximity with the control (37 mmol). The concomitant reduction in COD was maximum (15.61%) for 4 μmol Ni²⁺/L, followed by 15.33% for 60 μmol Fe²⁺/L, and the least for 6 mmol Mg²⁺/L (14.5%). The observations suggest the role of Fe²⁺ and Ni²⁺ in regulation of nitrogenase and hydrogenase, while that of Mg²⁺ mainly in the biosynthesis of photopigment bacteriochlorophyll (Bchl).     

Bio-hydrogen production and the F0F1-ATPase activity of Rhodobacter sphaeroides: Effects of various heavy metal ions

Rhodobacter sphaeroides MDC 6521 isolated from Arzni mineral springs in Armenia is able to produce bio-hydrogen (H₂) in anaerobic conditions upon illumination in the presence of various metal ions. The significant aspect in regulation of H₂ production by these bacteria and its energetics is the requirement for F₀F₁-ATPase, the main membrane enzyme responsible for generation of proton motive force under anaerobic conditions. In order to determine the mediatory role of F₀F₁ in H₂ production, the effects of various metal ions (Mn²⁺, Mg²⁺, Fe²⁺, Ni²⁺, and Mo⁶⁺) on N,N′-dicyclohexylcarbodiimide inhibited ATPase activity of R. sphaeroides membrane vesicles were investigated. These ions in appropriate concentrations considerably enhanced H₂ production, which was not observed in the absence of Fe²⁺, indicate the requirement for Fe²⁺. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity. In the absence of Fe²⁺ inhibition (∼80%) of ATPase activity was observed, which was increased by addition of metal ions. A higher ATPase activity was detected in the presence of Fe²⁺ (80 μM) and Mo⁶⁺ (16 μM). These results indicate a relationship between the F₀F₁-ATPase activity and H₂ production that might be a significant pathway to provide novel evidence of a requirement for F₀F₁-ATPase in H₂ production by R. sphaeroides.     

Concentration-dependent effects of metronidazole,inhibiting nitrogenase,on hydrogen photoproduction and proton-translocating ATPase activity of Rhodobacter sphaeroides

Rhodobacter sphaeroides MDC6522 is able to produce hydrogen (H₂) during photofermentation. Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) has been shown to affect bacterial growth under anaerobic nitrogen-limited conditions in a concentration-dependent manner (within the range of 0.1–2 mM) by increasing lag phase duration and decreasing growth rate. The addition of metronidazole into the growth medium resulted in a delayed decrease of redox potential (E_h): by the addition of 0.1 mM metronidazole E_h decreased to −590 ± 25 mV, whereas in the presence of 2 mM E_h drop down was to −175 ± 15 mV. H₂ production during R. sphaeroides growth disappeared in the presence of metronidazole. By addition of 0.5 mM metronidazole H₂ yield was ∼8 fold lower in comparison with control; whereas the bacterium was unable to produce H₂ in the presence of 1–2 mM. The effects of metronidazole on nitrogenase-dependent H₂ production by R. sphaeroides might be explained by change of general photosynthetic electron transport with metronidazole as an alternative electron acceptor instead of nitrogenase. ATPase activity of membrane vesicles was determined with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F₀F₁-ATPase. It was revealed that DCCD-inhibited ATPase activity increased in the presence of metronidazole. It is possible that this effect may be resulted in either by direct affect of metronidazole on the F₀F₁-ATPase, or by its effect on E_h regulating ATPase activity.     

The effect of Ni,Fe and Mg concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53

In the present study, the effect of Ni²⁺ (0–10 μmol/l), Fe²⁺ (0–200 μmol/l) and Mg²⁺ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni²⁺ was able to enhance the hydrogen production rate and the hydrogen yield; Fe²⁺ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni²⁺ and Fe²⁺ at a higher concentration inhibited cell growth. When Ni²⁺ and Fe²⁺ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H₂/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg²⁺ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H₂/mol acetate, but it was favorable to cell growth.     

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.     

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.     

Hydrogen production from acid hydrolyzed molasses by the hydrogen overproducing Escherichia coli strain HD701 and subsequent use of the waste bacterial biomass for biosorption of Cd(II) and Zn(II)

This study was devoted to investigate production of hydrogen gas from acid hydrolyzed molasses by Escherichia coli HD701 and to explore the possible use of the waste bacterial biomass in biosorption technology. In variable substrate concentration experiments (1, 2.5, 5, 10 and 15 g L⁻¹), the highest cumulative hydrogen gas (570 ml H₂ L⁻¹) and formation rate (19 ml H₂ h⁻¹ L⁻¹) were obtained from 10 g L⁻¹ reducing sugars. However, the highest yield (132 ml H₂ g⁻¹ reducing sugars) was obtained at a moderate hydrogen formation rate (11 ml H₂ h⁻¹ L⁻¹) from 2.5 g L⁻¹ reducing sugars. Subsequent to H₂ production, the waste E. coli biomass was collected and its biosorption efficiency for Cd²⁺ and Zn²⁺ was investigated. The biosorption kinetics of both heavy metals fitted well with the pseudo second-order kinetic model. Based on the Langmuir biosorption isotherm, the maximum biosorption capacities (q_max) of E. coli waste biomass for Cd²⁺ and Zn²⁺ were 162.1 and 137.9 (mg/g), respectively. These q_max values are higher than those of many other previously studied biosorbents and were around three times more than that of aerobically grown E. coli. The FTIR spectra showed an appearance of strong peaks for the amine groups and an increase in the intensity of many other functional groups in the waste biomass of E. coli after hydrogen production in comparison to that of aerobically grown E. coli which explain the higher biosorption capacity for Cd²⁺ or Zn²⁺ by the waste biomass of E. coli after hydrogen production. These results indicate that E. coli waste biomass after hydrogen production can be efficiently used in biosorption technology. Interlinking such biotechnologies is potentially possible in future applications to reduce the cost of the biosorption technology and duplicate the benefits of biological H₂ production technology.     

Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor

The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of downcomer and riser (A_d/A_r) in range of 0.4–3.2 were considered. Maximum biomass concentration of 1.63 g L⁻¹ in 9 d under light intensity of 120 μE m⁻² s⁻¹ was observed at A_d/A_r of 1.6. The mixing time of the reactors was inversely proportional to A_d/A_r. Maximal H₂ production was found to be 1600 mL L⁻¹ upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L⁻¹), autoclave (600 mL L⁻¹) and HCl treatment (1230 mL L⁻¹). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L⁻¹ as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H₂ production.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

Function of glucose catabolic pathways in hydrogen production from glucose in Rhodobacter sphaeroides 6016

Purple non-sulfur (PNS) bacteria can convert volatile fatty acids into hydrogen with a high substrate conversion efficiency. However, when PNS bacteria utilize sugars as a carbon source, such as glucose and sucrose, the substrate conversion efficiency is relatively low. In order to investigate the contributions of the glucose catabolic pathways in Rhodobacter sphaeroides 6016 to its hydrogen production, the cfxA gene from the Embden–Meyerhof–Parnas (EMP) pathway, edd from the Entner–Doudoroff (ED) pathway, and kdg from the semi-phosphorylative ED bypass were knocked out to construct the mutant strains edd⁻, cfxA⁻, and kdg⁻, respectively. Additionally, two of these three genes were knocked out to construct the mutant strains kdg⁻edd⁻, kdg⁻cfxA⁻, and cfxA⁻edd⁻. Hydrogen productions by these mutant strains were compared to that of the wild type strain 6016 using 25 mM glucose as a carbon source. Compared to 6016, variations in hydrogen production and growth were detected in the edd mutant strains (kdg⁻edd⁻, cfxA⁻edd⁻, and edd⁻), while no obvious changes were detected in the others. Notably, the kdg⁻edd⁻ mutant did not produce hydrogen, and its maximum growth was 70% less than that of R. sphaeroides 6016. These results indicate that the ED pathway and semi-phosphorylative ED bypass have a governing impact on cell growth and hydrogen production from glucose in R. sphaeroides 6016. The potential synergistic function of the ED pathway and semi-phosphorylative ED bypass and the reasons for the low hydrogen yield from sugar carbon sources in R. sphaeroides 6016 are discussed.     

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.     

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H₂). CO-dependent H₂ production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H₂ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein⁻¹) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein⁻¹). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein⁻¹. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H₂ from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H₂ase for CO-dependent H₂ production.     

Hydrogen production from C1 compounds by a novel marine hyperthermophilic archaeon Thermococcus onnurineus NA1

A novel marine hyperthermophile, Thermococcus onnurineus NA1, was found to grow on C1 carbon compounds, such as formate and carbon monoxide (CO), and produce hydrogen (H₂). In the present study, the growth and H₂ production of NA1 were examined to determine its potential as H₂ producer. NA1 showed relatively high specific growth rates, 0.48 h⁻¹ and 0.40 h⁻¹ with CO (20%, v/v) and formate (100 mM), respectively, when cultivated in batch mode in a minimal salt medium fortified with 1.0 g L⁻¹ yeast extract. On the other hand, cell growth in both cases stopped at approximately 6 h and the final cell densities were extremely low at 18.2 and 12.1 mg protein L⁻¹ with CO and formate, respectively. The maximum final cell density could be improved greatly to 36.0 mg protein L⁻¹ by optimizing CO content (50%, v/v) and yeast extract concentration (4.0 g L⁻¹), but it was still very low. During the cell growth, formate and CO were used as energy source rather than carbon source. In the resting cell experiments, NA1 exhibited remarkably high H₂ production activities as 385.0 and 207.5 μmol mg protein⁻¹ h⁻¹ for CO and formate, respectively. When formate (100 mM) or CO (100%, v/v) was added repeatedly at 30–35 h intervals, NA1 showed consistent H₂ production for 3 cycles with a yield of approximately 1.0 mol H₂ mol⁻¹ for both CO and formate. This study suggests that T. onnurineus NA1 has a high H₂ production potential from formate or CO but a method for achieving a high cell density culture is needed.     

Optimization of molecular hydrogen production by Rhodobacter sphaeroides O.U.001 in the annular photobioreactor using response surface methodology

Photofermentative H₂ production at higher rate is desired to make H₂ viable as cheap energy carrier. The process is influenced by C/N composition, pH levels, temperature, light intensity etc. In this study, Rhodobacter sphaeroides strain O.U 001 was used in the annular photobioreactor with working volume 1 L, initial pH of 6.7 ± 0.2, inoculum age 36 h, inoculum volume 10% (v/v), 250 rpm stirring and light intensity of 15 ± 1.1 W m⁻². The effect of parameters, i.e. variation in concentration of DL malic acid, L glutamic acid and temperature on the H₂ production was noted using three factor three level full factorial designs. Surface and contour plots of the regression models revealed optimum H₂ production rate of 7.97 mL H₂ L⁻¹ h⁻¹ at 32 °C with 2.012 g L⁻¹ DL malic acid and 0.297 g L⁻¹ L glutamic acid, which showed an excellent correlation (99.36%) with experimental H₂ production rate of 7.92 mL H₂ L⁻¹ h⁻¹.     

Novel Ni–ZrO2 catalyst doped with Yb2O3 for ethanol steam reforming

A type of Yb₂O₃ doped Ni–ZrO₂ catalyst for ethanol steam reforming was developed, and displayed excellent catalyzing performance for the selective formation of H₂ and CO₂. Over a Ni_1.25Zr₁Yb_0.8 catalyst, STY(H₂) can maintain stable at the level of 0.396 mol h⁻¹ g⁻¹ (data taken 120 h after the reaction started) under the reaction conditions of 0.5 MPa and 723 K, which was 1.6 times that (0.247 mol h⁻¹ g⁻¹) of the Yb-free counterpart Ni_1.25Zr₁. Characterization of the catalyst revealed that dissolution of an appropriate amount of Yb³⁺ ions in the zirconia host resulted in the formation of the Zr–Yb composite oxide with cubic-ZrO₂ structure, c-(Zr–Yb)O_z, which inhibited effectively the transformation of c-ZrO₂ to thermodynamically more stable m-ZrO₂, thus avoiding sintering of the (Zr–Yb)O_z composite. It was demonstrated that the doping of Yb₂O₃ to Ni–ZrO₂ changed also the valence states or the micro-environments of the Ni-species at the quasi-active surface of the tested catalyst, which was conducive to inhibiting agglomeration of the Ni_x⁰–Niⁿ⁺ species active catalytically, with resulting in maintaining the high metallic nickel dispersion and inhibiting coking. The aforementioned two factors both contributed to improving the activity and operating stability as well as heat-resistant quality of the catalyst.