Remarkable enhancement on hydrogen production performance of Rhodobacter sphaeroides by disrupting spbA and hupSL genes

The redox balance and bacteriochlorophyll (Bchl) synthesis are both significant to hydrogen generation in photosynthetic bacteria. In this study, spbA and hupSL genes were knocked out from the genome of Rhodobacter sphaeroides HY01. The UV–vis spectra showed that the Bchl contents of spbA mutants were enhanced under photosynthetic conditions. The hydrogen yields of WH04 (hupSL⁻) and WSH10 (spbA⁻, hupSL⁻) mutants increased by 19.4%, 21.8%, and the maximum hydrogen evolution rates increased by 29.9% and 55.0% respectively using glutamate as sole nitrogen source. The maximum hydrogen production rate of WSH10 was up to 141.9 mL/(L·h). The nifH expression levels of the mutants and the wild type supported the correlation between hydrogen production and nitrogenase activity. The results demonstrate that disruption of spbA in R. sphaeroides can partially derepress the ammonium inhibition in nitrogenase activity, and indicate that spbA is a negative regulator in nitrogenase synthesis in the presence of ammonium.     

Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp

Hydrogen gas production from sugar solution derived from acid hydrolysis of ground wheat starch by photo-fermentation was investigated. Three different pure strains of Rhodobacter sphaeroides (RV, NRLL and DSZM) were used in batch experiments to select the most suitable strain. The ground wheat was hydrolyzed in acid solution at pH = 3 and 90 °C in an autoclave for 15 min. The resulting sugar solution was used for hydrogen production by photo-fermentation after neutralization and nutrient addition. R. sphaeroides RV resulted in the highest cumulative hydrogen gas formation (178 ml), hydrogen yield (1.23 mol H₂ mol⁻¹ glucose) and specific hydrogen production rate (46 ml H₂ g⁻¹ biomass h⁻¹) at 5 g l⁻¹ initial total sugar concentration among the other pure cultures. Effects of initial sugar concentration on photo-fermentation performance were investigated by varying sugar concentration between 2.2 and 13 g l⁻¹ using the pure culture of R. sphaeroides RV. Cumulative hydrogen volume increased from 30 to 232 ml when total sugar concentration was increased from 2.2 to 8.5 g l⁻¹. Further increases in initial sugar concentration resulted in decreases in cumulative hydrogen formation. The highest hydrogen formation rate (3.69 ml h⁻¹) and yield (1.23 mol H₂ mol⁻¹ glucose) were obtained at a sugar concentration of 5 g l⁻¹.     

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.     

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.     

Metabolic engineering of Escherichia coli strains for co-production of hydrogen and ethanol from glucose

The co-production of H₂ and ethanol from glucose was studied to address the low H₂ production yield in dark fermentation. Several mutant strains devoid of ackA-pta, pfkA or pgi were developed using Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB as base strain. Disruption of ackA-pta eliminated acetate production during glucose fermentation but resulted in the secretion of a significant amount of pyruvate (0.73 mol mol⁻¹ glucose) without improving the co-production of H₂ and ethanol. When pfkA or pgi was further disrupted to enhance NAD(P)H supply by diverting the carbon flux from Embden-Meyerhof-Parnas (EMP) pathway to the pentose phosphate pathway (PPP), the cell growth of both strains was severely impaired under anaerobic conditions, and only the ΔpfkA mutant could recover its growth after adaptive evolution. The production yields of the ΔpfkA strain (H_2, 1.03 mol mol⁻¹ glucose and ethanol, 1.04 mol mol⁻¹ glucose) were higher than those of the pfkA⁺ strain (H₂, 0.69 mol mol⁻¹ glucose and ethanol, 0.95 mol mol⁻¹ glucose), but pyruvate excretion was not reduced. The excessive excretion of pyruvate in the ΔpfkA mutant was attributed to an insufficient NAD(P)H supply caused by the diversion of carbon flux from the EMP pathway to the Entner-Doudoroff pathway (EDP), rather than the PPP as intended. This study suggests that co-production of H₂ and ethanol from glucose is possible, but further metabolic pathway engineering is required to fully activate PPP under anaerobic conditions.     

Relationship between molecular hydrogen production,proton transport and the F0F1-ATPase activity in Rhodobacter sphaeroides strains from mineral springs

This article aims to study hydrogen production and proton transport in two strains of purple non-sulfur bacterium Rhodobacter sphaeroides isolated from mineral springs of Armenia. This bacterium is able to grow and produce molecular hydrogen (H₂) in anaerobic conditions upon illumination. Along with H₂ production, a marked decrease in redox potential and the alkalization of the medium have been observed; the latter might be the evidence of proton influx. H₂ production and alkalization of the medium by whole cells both are suppressed by the F₀F₁-ATPase inhibitors – N,N′-dicyclohexylcarbodiimide (DCCD), sodium azide (NaN₃) and protonophore – carbonyl cyanide m-chlorophenylhydrazone (CCCP). Membrane vesicles of two strains of R. sphaeroides demonstrate ATPase activity, inhibited by DCCD and NaN₃, but not by CCCP. These results indicate a relationship between H₂ production, proton transport and the F₀F₁-ATPase activity that might be a pathway to regulate bacterial activity under anaerobic conditions.     

Optimization of thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

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.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

Hydrogen productivity of photosynthetic bacteria on dark fermenter effluent of potato steam peels hydrolysate

Hydrogen productivities of different photosynthetic bacteria have been searched on real thermophilic dark fermentation effluents (DFE). The results obtained with potato steam peels hydrolysate (PSP) DFE were compared to glucose DFE. Photobiological hydrogen production has been carried out in indoor, batch photobioreactors using several strains of purple non-sulfur (PNS) bacteria such as Rhodobacter capsulatus (DSM1710), Rhodobacter capsulatus hup- (YO3), Rhodobacter sphaeroides O.U.001 (DSM5864), Rb. sphaeroides O.U.001 hup- and Rhodopseudomonas palustris.The efficiency of photofermentation depends highly on the composition of the effluent and the PNS bacterial strain used. Rb. sphaeroides produced the highest amount of hydrogen on glucose DFE. Rb. capsulatus gave better results on PSP DFE. This study demonstrates that photobiological hydrogen production with high efficiency and productivity is possible on thermophilic dark fermentation effluents. Consequently, a sequential operation of dark fermentation and photofermentation is a promising route to produce hydrogen, and it provides a higher hydrogen yield compared to single step processes.     

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.     

Cloning and knockout of formate hydrogen lyase and H2-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A (△hycA), IAM1183-O (△hybO), and IAM1183-AO (△hycA/△hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 ± 38.59, 2747.203 ± 13.25 and 3372.019 ± 4.39 (ml h⁻¹ g⁻¹dry cell weight), respectively, higher than that of the wild strain (2321.861 ± 15.34 ml h⁻¹ g⁻¹dry cell weight). The total H₂ yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H₂/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H₂/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183△hycA/△hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L.     

The effects of seed sludge and hydraulic retention time on the production of hydrogen from a cassava processing wastewater and glucose mixture in an anaerobic fluidized bed reactor

The potential for co-fermentation of a cassava processing wastewater and glucose mixture was studied in anaerobic fluidized bed reactors. The effects of different hydraulic retention times (HRTs) (10–2 h) and varying sources of inoculum are reported. The sludge from a UASB reactor that had been used to treat poultry slaughterhouse wastewater (SP) resulted in the highest yields of hydrogen (HY) and ethanol (EtOHY) of 1.0 mmol H₂ g⁻¹ COD (10 h) and 3.0 mmol EtOH g⁻¹ COD (6 h). The sludge from a UASB reactor used for the treatment of swine wastewater (SW) resulted in a maximum HY of 0.65 mmol H₂ g⁻¹ COD (6 h) and EtOHY of 2.1 mmol g⁻¹ COD (10 and 8 h). Methane was produced with a maximum production of 9.68 L CH₄ d⁻¹ L⁻¹. Based on phylogenetic analysis of 16S rRNA, bacteria and methanogenic archaea similar to Lactobacillus and Methanobacterium, respectively, were identified.     

Effects of light source,intensity and lighting regime on bio-hydrogen production from ground wheat starch by combined dark and photo-fermentations

Combined dark and photo-fermentation of ground wheat starch was carried out by using different light sources, intensities and lighting regime. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides-RV with a certain light/dark bacteria ratio was used in batch experiments. Tungsten, fluorescent, infrared (IR), tungsten + infrared, halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (178 ml) and yield (218 ml g⁻¹ starch). Combined fermentations were performed at different light intensities (1–10 klux) using the halogen lamp in the second set of experiments. The optimum light intensity was found to be 10 klux (approx. 352 Wm⁻²) resulting in the highest cumulative hydrogen (111 ml) and hydrogen yield (139 ml H₂ g⁻¹ starch). Hydrogen formation was limited by the availability of light at low light intensities below 10 klux. Durations of dark/light cycles were changed to determine the most suitable lighting regime. Hydrogen gas formation increased with increasing cycle time and continuous lighting resulted in the highest cumulative hydrogen formation and hydrogen yield.     

Developing a thermophilic hydrogen-producing co-culture for efficient utilization of mixed sugars

Previous studies on the extreme thermophile Caldicellulosiruptor saccharolyticus revealed that the organism produces high yields of hydrogen on glucose and xylose, the major components of lignocellulosic hydrolysates. Preliminary experiments on mixed sugar substrates, however, indicated that xylose was preferred over glucose. The sugar preference of some other extreme thermophiles, including Caldicellulosiruptor owensensis, Caldicellulosiruptor kristjanssonii and newly enriched, thermophilic compost sludge microflora, was investigated in an attempt to find complementary organisms to C. saccharolyticus for rapid and efficient utilization of lignocellulosic sugars. The behavior of C. owensensis and C. kristjanssonii appeared to be similar to that of C. saccharolyticus, either in pure cultures or in co-cultures with the latter. Co-culturing C. saccharolyticus with the enriched compost microflora resulted in fast, simultaneous consumption of both glucose and xylose in the medium with a relatively high specific hydrogen production rate, 40 mmol (gCDW)⁻¹ h⁻¹, and high volumetric productivity, 22.5 mmol l⁻¹ h⁻¹.     

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.     

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H₂ is rapidly consumed by the uptake hydrogenase.This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL⁻ (deficient in the uptake hydrogenase), hoxH⁻ (deficient in the bidirectional hydrogenase) and hupL⁻/hoxH⁻ (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N₂ and/or CO₂ addition), light intensity (54 and 152 ??Em⁻²s⁻¹), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium.In every assay, the hupL⁻ and hupL⁻/hoxH⁻ mutants stood out over wild-type cells and the hoxH⁻ mutant. Nevertheless, the hupL⁻ mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 ??Em⁻²s⁻¹ in the light period, with 1 ??M of NiCl₂ supplementation in the culture medium, and under a propane atmosphere.In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO₂ addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO₂ plus 1% of N₂ did not affect positively hydrogen production rate.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of ground wheat solution: Effects of light source and light intensity

Dark fermentation effluent of wheat powder solution was subjected to light fermentation for bio-hydrogen production using different light sources and intensities. Tungsten, fluorescent, infrared (IR), halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Pure culture of Rhodobacter sphaeroides-RV was used in batch light fermentation experiments. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (CHF, 252 ml) and yield (781 ml H₂ g⁻¹ TVFA). In the second set of experiments, light fermentations were performed at different light intensities (1–10 klux) using halogen lamp. The optimum light intensity was found to be 5 klux (approx. 176 Wm⁻²) resulting in the highest CHF (88 ml) and hydrogen yield (1037 ml H₂ g⁻¹TVFA). Hydrogen formation was limited by the availability of light at low light intensities below 5 klux and was inhibited by the excess light above 5 klux.