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.     

Improvement of photo-fermentative hydrogen production performance by expressing tetracycline resistance genes tetAR in Rhodobacter sphaeroides HY01

A typical expression plasmid, pRK415, was widely used to introduce cloned DNA fragment into a broad range of Gram-negative bacteria, including Rhodobacter sphaeroides. Tetracycline (Tc) is required for stable inheritance of the plasmid, but it is subject to photooxidation, inhibitory to the growth of the host strain. However, in this study, the introduction of pRK415 into R. sphaeroides HY01 under tetracycline stress showed clear enhancement on H₂ production performance of the recombinant strain pRK415/HY01. The maximum H₂ production rate of pRK415/HY01 was enhanced by 10%–70% compared with the control group in the absent of Tc. Supplementing with Tc, the wild type strain HY01 showed repressed cell growth and reduced H₂ production performance. The tetAR genes knockout test demonstrated that the expression of tetAR genes on pRK415 promoted the H₂ production performance. And within tolerance concentration (Tc < 2.5 mg/L), higher Tc concentration led to higher H₂ production performance of pRK415/HY01. Expression of tetAR genes in the genome of R.sphaeroides HY01 by substituting partial of hupSL genes enhanced the H₂ production performance as well. The mechanism of the H₂ production performance enhancement was discussed.     

Dilute-acid pretreatment of barley straw for biological hydrogen production using Caldicellulosiruptor saccharolyticus

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H₃PO₄ and H₂SO₄, and to a lesser extent, HNO₃. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.     

Thermophilic biohydrogen production from energy plants by Caldicellulosiruptor saccharolyticus and comparison with related studies

Air-dried samples of sweet sorghum, sugarcane bagasse, wheat straw, maize leaves and silphium were utilized without chemical pretreatment as sole energy and carbon sources for H₂ production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus. The specific H₂ production rates and yields were determined in the batch fermentation process. The best substrate was wheat straw, with H₂ production capacity of 44.7 L H₂ (kg dry biomass)^?1 and H₂ yield of 3.8 mol H₂ (mol glucose)^?1. Enzymatically pretreated maize leaves exhibited H₂ production of 38 L H₂ (kg dry biomass)^?1. Slightly less H₂ was obtained from homogenized whole plants of sweet sorghum. Sweet sorghum juice was an excellent H₂ source. Silphium trifoliatum was also fermented though with a moderate production. The results showed that drying is a good storage method and raw plant biomass can be utilized efficiently for thermophilic H₂ production. The data were critically compared with recently published observations.     

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.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

Assessment of the adequacy of different Mediterranean waste biomass types for fermentative hydrogen production and the particular advantage of carob (Ceratonia siliqua L.) pulp

The conversion of agro-industrial byproducts, residues and microalgae, which are representative or adapted to the Mediterranean climate, to hydrogen (H₂) by C. butyricum was compared. Five biomass types were selected: brewery’s spent grain (BSG), corn cobs (CC), carob pulp (CP), Spirogyra sp. (SP) and wheat straw (WS). The biomasses were delignified and/or saccharified, except for CP which was simply submitted to aqueous extraction, to obtain fermentable solutions with 56.2–168.4 g total sugars L^?1. In small-scale comparative assays, the H₂ production from SP, WS, CC, BSG and CP reached 37.3, 82.6, 126.5, 175.7 and 215.8 mL (g biomass)^?1, respectively. The best fermentable substrate (CP) was tested in a pH-controlled batch fermentation. The H₂ production rate was 204 mL (L h)^?1 and a cumulative value of 3.9 L H₂ L^?1 was achieved, corresponding to a H₂ production yield of 70.0 mL (g biomass)^?1 or 1.6 mol (mol of glucose equivalents)^?1_. The experimental data were used to foresight a potential energy generation of 2.4 GWh per year in Portugal, from the use of CP as substrate for H₂ production.     

Improvement of hydrogen production by metabolic engineering of Escherichia coli: Modification on both the PTS system and central carbon metabolism

Lignocellulosic-based production of bio-hydrogen (H₂) by Escherichia coli requires efficient consumption of pentoses and hexoses. However, carbon catabolite repression (CCR) causes sequential utilization of carbohydrates and in some cases null consumption of less preferred carbohydrates, such as xylose. In this work, we evaluated the effect of elimination of the phosphotransferase system (PTS), responsible for CCR in strain E. coli WDH (ΔhycA) on H₂ production using mixtures of glucose-xylose as carbon source. Elimination of ptsG gene (glucose permease-enzyme IIB), allowed simultaneous consumption of glucose and xylose, and improved H₂ production 1.2-times with respect to the parenteral strain. Whereas, elimination of ptsG gene in combination with deletion of ldhA (d-lactate dehydrogenase) and/or frdD (fumarate reductase) genes, improved H₂ production 2.5-times with a H₂ yield of 0.27 mol·C-mol⁻¹, using mixtures of glucose/xylose or wheat straw hydrolysate. Interestingly, besides the improvement on H₂ production, E. coli WDH-GFA (ΔhycA, ΔptsG, ΔfrdD, ΔldhA) strain also produced ethanol as the main carbon by-product. These results show that elimination of ptsG, in combination with a modified central carbon metabolism improves the production of H₂.     

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h^?1 and the average pseudo-steady state hydrogen production rate was 36 mL H₂ L^?1 medium h^?1. The cumulative hydrogen production was 430 mL H₂ L^?1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H₂ L^?1 h^?1 and the cumulative hydrogen production was 517 mL H₂ L^?1 medium. Reducing the feed rate to less than 48 mL h^?1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.     

Producing hydrogen from the fermentation of cheese whey and glycerol as cosubstrates in an anaerobic fluidized bed reactor

This study aimed to evaluate the effect of the organic loading rate (OLR) (60, 90, and 120 g Chemical Oxygen Demand (COD). L^?1. d^?1) on hydrogen production from cheese whey and glycerol fermentation as cosubstrates (50% cheese whey and 50% glycerol on a COD basis) in a thermophilic fluidized bed reactor (55 °C). The increase in the OLR to 90 gCOD.L^?1. d^?1 favored the hydrogen production rate (HPR) (3.9 L H₂. L^?1. d^?1) and hydrogen yield (HY) (1.7 mmol H₂. gCOD^?1_app) concomitant with the production of butyric and acetic acids. Employing 16S rRNA gene sequencing, the highest hydrogen production was related to the detection of Thermoanaerobacterium (34.9%), Pseudomonas (14.5%), and Clostridium (4.7%). Conversely, at 120 gCOD.L^?1. d^?1, HPR and HY decreased to 2.5 L H₂. L^?1. d^?1 and 0.8 mmol H₂. gCOD^?1_app, respectively, due to lactic acid production that was related to the genera Thermoanaerobacterium (50.91%) and Tumebacillus (23.56%). Cofermentation favored hydrogen production at higher OLRs than cheese whey single fermentation.     

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.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Genetically engineered deletion in the N-terminal region of nifA1 in R. capsulatus to enhance hydrogen production

NifA is the primary activator of nitrogenase, and the N-terminal domain of nifA is sensitive to ammonium concentration. In this work, a mutant Rhodobacter capsulatus ZX01 with a genetically engineered deletion in the N-terminal region of nifA1 was constructed by employing overlap extension PCR to mitigate the inhibition of ammonium on nitrogenase expression in photosynthetic bacteria. The effects of different ammonium ion concentrations on the growth and photo-fermentative hydrogen production performance of wild-type strain R. capsulatus SB1003 and mutant ZX01 with glucose and volatile fatty acids as the carbon sources were studied, respectively. When the ratio of NH₄⁺-N was 20% and 30%, the hydrogen yield of the mutant ZX01 was enhanced by 14.8% and 20.9% compared with that of R. capsulatus SB1003 using 25 mM acetic acid and 34 mM butyric acid as the carbon source, respectively. In comparison, using 30 mM glucose as the carbon source, the hydrogen yield of ZX01 was increased by 17.7% and 22.2% compared with that of R. capsulatus SB1003 when the ratio of NH₄⁺-N was 20% and 30%, and the nitrogenase activity of ZX01 was also enhanced by 38.0% and 47.6%, respectively. When using 10 mM NH₄⁺ as a single nitrogen source, ZX01 showed a 2.6-fold increase in H₂ production. These results indicated that ZX01 demonstrated higher ammonium tolerance and better hydrogen production performance than the wild-type. The deletion in the N-terminal region of nifA1 could partially de-repress the nitrogenase activity inhibited by ammonium.     

Biohydrogen production from diluted-acid hydrolysate of rice straw in a continuous anaerobic packed bed biofilm reactor

Bio-hydrogen production in a continuously operated anaerobic packed bed biofilm reactor (APBR) using acid-hydrolysate of rice straw as feedstock and inoculated with an anaerobic mesophilic sludge from a municipal wastewater treatment plant was investigated at three different HRTs (17, 8.2 and 2 h). Fermentable sugars solution achieved from a two-stage diluted acid hydrolysis of rice straw was used as the feedstock. First, rice straw was treated with 1% w v^?1 sulfuric acid at 120 °C for 30 min with a yield of 58.5% xylose. Higher temperature of 180 °C for 10 min at 0.5% w v^?1 sulfuric acid was applied in the second stage in which cellulosic crystalline structure was partially depolymerized to glucose with a yield of 19.3% glucose. Hydrogen production rate and yield were enhanced as the hydraulic retention time was decreased with a maximum production rate of 252 mL L^?1 h^?1 and yield of 1 mol H₂ mol^?1 sugar consumed at 2 h HRT. Experimental results illustrated the increase of COD conversion from 44% to 47% by shortening the HRT from 17 to 2 h. Furthermore, acetic acid and butyric acid production were reduced slower than other soluble metabolites like ethanol.     

Factors affecting hydrogen production from rice straw wastes in a mesophillic up-flow anaerobic staged reactor

Anaerobic biodegradation of rice straw wastes for H₂ production via mesophillic up-flow anaerobic staged reactor (UASR) was investigated. Two batch experiments were carried out. The 1st experiment was conducted to assess the effect of pre-acidification process on H₂ production rate (R_m). The results showed that the maximum R_m of 136.64 mlH₂/h was achieved for pre-acidified (0.72%) rice straw waste, which was approximately 28.64-fold greater than that in untreated rice straw. The H₂ content in the biogas was 52.0% and there was no significant methane observed in this study. The pre-acidified rice straw was used for subsequent experiments concerning the influences of environmental factors such as pH, contact time, and substrate concentration on H₂ yield (HY). Trends indicate that both high and low-end pH is unfavorable and substrate concentration of 30 g COD/l and contact time of 30 h is recommended for H₂ production from pre-acidified rice straw.     

Global modeling of hydrogen using GFDL-AM4.1: Sensitivity of soil removal and radiative forcing

Hydrogen (H₂) has been proposed as an alternative energy carrier to reduce the carbon footprint and associated radiative forcing of the current energy system. Here, we describe the representation of H₂ in the GFDL-AM4.1 model including updated emission inventories and improved representation of H₂ soil removal, the dominant sink of H₂. The model best captures the overall distribution of surface H₂, including regional contrasts between climate zones, when v_d(H₂) is modulated by soil moisture, temperature, and soil carbon content. We estimate that the soil removal of H₂ increases with warming (2–4% per K), with large uncertainties stemming from different regional response of soil moisture and soil carbon. We estimate that H₂ causes an indirect radiative forcing of 0.84 mW m^?2/(Tg(H₂)yr^?1) or 0.13 mW m^?2 ppbv^?1, primarily due to increasing CH₄ lifetime and stratospheric water vapor production.     

Design of a novel biohythane process with high H2 and CH4 production rates

A biohythane process based on wheat straw including: i) pretreatment, ii) H₂ production using Caldicellulosiruptor saccharolyticus, iii) CH₄ production using an undefined consortium, and iv) gas upgrading using an amine solution, was assessed through process modelling including cost and energy analysis. According to simulations, a biohythane gas with the composition 46–57% H₂, 43–54% CH₄ and 0.4% CO₂, could be produced at high production rates (2.8–6.1 L/L/d), with 93% chemical oxygen demand (COD) reduction, and a net energy yield of 7.4–7.7 kJ/g dry straw. The model was calibrated and verified using experimental data from dark fermentation (DF) of wheat straw hydrolysate, and anaerobic digestion of DF effluent. In addition, the effect of gas recirculation was investigated by both wet experiments and simulation. Sparging improved H₂ productivities and yields, but negatively affected the net energy gain and cost of the overall process.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Evidence for hydrogenase-4 catalyzed biohydrogen production in Escherichia coli

Biohydrogen production by Escherichia coli during fermentation of the mixture of glycerol, glucose and formate at different pH values was studied. Employing mutants lacking large subunits of different hydrogenases (Hyd), it was reported that, at pH 7.5, H₂ production was produced except in a hyaB hybC hycE triple mutant, thus suggesting compensatory H₂-producing functions of the Hyd enzymes. Activity of Hyd-4 was revealed in glucose assays at pH 7.5 in the triple mutant whereby 62% of the wild type level of H₂ production was derived from Hyd-4. In formate assays, it was shown, that, first, the hyaB hybC double mutant had a H₂ production ～3 fold higher than wild type, indicating that Hyd-1 and Hyd-2 oxidize H₂, and second, that at pH 5.5, Hyd-4 and Hyd-3 were responsible for H₂ production. These findings are significant when applying various carbon sources such as sugars, alcohol and organic acids for biohydrogen production.     

Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates

Acetone butanol ethanol (ABE) was produced from hydrolysed corn stover and switchgrass using Clostridium beijerinckii P260. A control experiment using glucose resulted in the production of 21.06 g L^?1 total ABE. In this experiment an ABE yield and productivity of 0.41 and 0.31 g L^?1 h^?1 was achieved, respectively. Fermentation of untreated corn stover hydrolysate (CSH) exhibited no growth and no ABE production; however, upon dilution with water (two fold) and wheat straw hydrolysate (WSH, ratio 1:1), 16.00 and 18.04 g L^?1 ABE was produced, respectively. These experiments resulted in ABE productivity of 0.17–0.21 g L^?1 h^?1. Inhibitors present in CSH were removed by treating the hydrolysate with Ca(OH)₂ (overliming). The culture was able to produce 26.27 g L^?1 ABE after inhibitor removal. Untreated switchgrass hydrolysate (SGH) was poorly fermented and the culture did not produce more than 1.48 g L^?1 ABE which was improved to 14.61 g L^?1. It is suggested that biomass pretreatment methods that do not generate inhibitors be investigated. Alternately, cultures resistant to inhibitors and able to produce butanol at high concentrations may be another approach to improve the current process.