Photo-fermentative H2 Production Research Articles

Construction of a Rhodobacter sphaeroides Strain That Efficiently Produces Hydrogen Gas from Acetate without Poly(β-Hydroxybutyrate) Accumulation: Insight into the Role of PhaR in Acetate Metabolism.

The purple nonsulfur phototrophic bacterium Rhodobacter sphaeroides produces hydrogen gas (H2) from acetate. An approach to improve the H2 production is preventing accumulation of an intracellular energy storage molecule known as poly(β-hydroxybutyrate) (PHB), which competes with H2 production for reducing power. However, disruption of PHB biosynthesis has been reported to severely impair the acetate assimilation depending on the genetic backgrounds and/or culture conditions. To solve this problem, we analyzed the relationship between PHB accumulation and acetate metabolism in R. sphaeroides. Gene deletion analyses based on the wild-type strain revealed that among the two polyhydroxyalkanoate synthase genes in the genome, phaC1, but not phaC2, is essential for PHB accumulation, and the phaC1 deletion mutant exhibited slow growth with acetate. On the other hand, a strain with the deletion of phaC1 together with phaR, which encodes a transcriptional regulator capable of sensing PHB accumulation, exhibited growth comparable to that of the wild-type strain despite no accumulation of PHB. These results suggest that PHB accumulation is required for normal growth with acetate by altering the expression of genes under the control of phaR. This hypothesis was supported by a transcriptome sequencing (RNA-seq) analysis revealing that phaR is involved in the regulation of the ethylmalonyl coenzyme A pathway for acetate assimilation. Consistent with these findings, deletion of phaC1 in a genetically engineered H2-producing strain resulted in lower H2 production from acetate due to growth defects, whereas deletion of phaR together with phaC1 restored growth with acetate and increased H2 production from acetate without PHB accumulation. IMPORTANCE This study provides a novel approach for increasing the yield of photofermentative H2 production from acetate by purple nonsulfur phototrophic bacteria. This study further suggests that polyhydroxyalkanoate is not only a storage substance for carbon and energy in bacteria, but may also act as a signaling molecule that mediates bacterial metabolic adaptations to specific environments. This notion will be helpful for understanding the physiology of polyhydroxyalkanoate-producing bacteria, as well as for their metabolic engineering via synthetic biology.

Promoting photo-fermentative hydrogen production performance by substituting the rnf promoter in Rhodobacter capsulatus

The Rnf (Rhodobacter nitrogen fixation) complex encoded by rnfABCDGEH genes is a crucial enzyme for the functioning of nitrogenase. In this work, we studied the effect of Rnf complex expression level on hydrogen production in the hydrogen-producing bacterium Rhodobacter capsulatus SB 1003 by expressing rnf genes with three autologous promoters. Substitution of the rnf promoter with that of pucB resulted in a 1.59-fold increase in rnf transcription level and 1.3-fold increase in nitrogenase activity. Under various test conditions, maximum H2 yields and production rates were increased by 12.4%–24.7% and 6.5%–22.9%, respectively. There was a further increase in H2 production when the Prnf::PpucB::rnf fusion construct was expressed in a background strain with both the alternative Fe nitrogenase and the Mo nitrogenase co-expressed. The results demonstrate that the Rnf complex catalyzes a rate-limiting step in photo-fermentative H2 production. The mutants with overexpressed Rnf complex are more competitive than naturally isolated strains under low light condition or in scale-up bioreactors.

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.

Introduction of Glyoxylate Bypass Increases Hydrogen Gas Yield from Acetate and l-Glutamate in Rhodobacter sphaeroides.

Rhodobacter sphaeroides produces hydrogen gas (H2) from organic compounds via nitrogenase under anaerobic-light conditions in the presence of poor nitrogen sources, such as l-glutamate. R. sphaeroides utilizes the ethylmalonyl-coenzyme A (EMC) pathway for acetate assimilation, but its H2 yield from acetate in the presence of l-glutamate has been reported to be low. In this study, the deletion of ccr encoding crotonyl-coenzyme A (crotonyl-CoA) carboxylase/reductase, a key enzyme for the EMC pathway in R. sphaeroides, revealed that the EMC pathway is essential for H2 production from acetate and l-glutamate but not for growth and acetate consumption in the presence of l-glutamate. We introduced a plasmid expressing aceBA from Rhodobacter capsulatus encoding two key enzymes for the glyoxylate bypass into R. sphaeroides, which resulted in a 64% increase in H2 production. However, compared with the wild-type strain expressing heterologous aceBA genes, the strain with aceBA introduced in the genetic background of an EMC pathway-disrupted mutant showed a lower H2 yield. These results indicate that a combination of the endogenous EMC pathway and a heterologously expressed glyoxylate bypass is beneficial for H2 production. In addition, introduction of the glyoxylate bypass into a polyhydroxybutyrate (PHB) biosynthesis-disrupted mutant resulted in a delay in growth along with H2 production, although its H2 yield was comparable to that of the wild-type strain expressing heterologous aceBA genes. These results suggest that PHB production is important for fitness to the culture during H2 production from acetate and l-glutamate when both acetate-assimilating pathways are present.IMPORTANCE As an alternative to fossil fuel, H2 is a promising renewable energy source. Although photofermentative H2 production from acetate is key to developing an efficient process of biohydrogen production from biomass-derived sugars, H2 yields from acetate and l-glutamate by R. sphaeroides have been reported to be low. In this study, we observed that in addition to the endogenous EMC pathway, heterologous expression of the glyoxylate bypass in R. sphaeroides markedly increased H2 yields from acetate and l-glutamate. Therefore, this study provides a novel strategy for improving H2 yields from acetate in the presence of l-glutamate and contributes to a clear understanding of acetate metabolism in R. sphaeroides during photofermentative H2 production.

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.

Novel properties of photofermentative biohydrogen production by purple bacteria Rhodobacter sphaeroides: effects of protonophores and inhibitors of responsible enzymes.

BackgroundBiohydrogen (H2) production by purple bacteria during photofermentation is a very promising way among biological H2 production methods. The effects of protonophores, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol (DNP), and inhibitors of enzymes, involved in H2 metabolism, metronidazole (Met), diphenyleneiodonium (DPI), and dimethylsulphoxide (DMSO) on H2 production by Rhodobacter sphaeroides MDC6522 isolated from Jermuk mineral springs in Armenia have been investigated in both nitrogen-limited and nitrogen-excess conditions.ResultsWith the increase of inhibitors concentrations H2 yield gradually decreased. The complete inhibition of H2 production was observed in the presence of DPI and CCCP. DPI’s solvent—DMSO in low concentration did not significantly affect H2 yield. N,N′-dicyclohexylcarbodiimide (DCCD)-inhibited the FOF1-ATPase activity of bacterial membrane vesicles was analyzed in the presence of inhibitors. Low concentrations of DPI and DMSO did not affect ATPase activity, whereas Met and CCCP stimulated enzyme activity. The effect of DNP was similar to CCCP.Conclusions and significanceThe results have shown the low concentration or concentration dependent effects of protonophores and nitrogenase and hydrogenase inhibitors on photofermentative H2 production by Rh. sphaeroides in nitrogen-limited and nitrogen-excess conditions. They would be significant to understand novel properties in relationship between nitrogenase, hydrogenase and the FOF1-ATPase in Rh. sphaeroides, and regulatory pathways of photofermentation. The inhibitors of nitrogenase and hydrogenase can be used in biotechnology for regulation of H2 production in different technology conditions and development of scale-up applications, for biomass and energy production using purple bacterial cells.

Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis

The effect of a two-step cornstalk pretreating process, NaOH delignification followed by enzymolysis with cellulase and hemicellase, on dark- and photo-fermentative H2 production was studied. A five-factor and five-level orthogonal experimental array was designed and conducted to study the effect of NaOH concentration (0–1%), hydrolysis time (0–3 h), hydrolysis temperature (98–126 °C), cellulase (0–18 IU/g-cornstalk) and hemicellulase dosage (0–2400 IU/g-cornstalk) on pretreatment efficiency determined by H2 production. With NaOH 0.75%, hydrolysis temperature 108 °C, hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemicellulase 2400 IU/g-CS, a maximum reducing sugar yield of 0.56 ± 0.03 g/g-CS and maximum H2 yield of 163.1 mL-H2/g-cornstalk were obtained under dark-fermentation, and a maximum H2 yield of 339.5 mL-H2/g-cornstalk was obtained under photo-fermentation. According to the results, the significance of the five parameters on H2 production was listed in high-to-low order as: NaOH concentration, cellulase dosage, hydrolysis temperature, hemicellulase and hydrolysis time. The effect of the alkaline delignification on pretreatment efficiency and the future efforts on improving H2 production from agricultural wastes were also discussed.

Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers

Photo-fermentative production of hydrogen (H2) by purple bacterium Rhodobacter sphaeroides MDC6521 from Armenian mineral springs and its regulation by external reducers and oxidizers have been investigated. Reducers such as dl-dithiothreitol (DTT) and dithionite suppress bacterial growth but enhance H2 yield of R. sphaeroides, whereas oxidizer ferricyanide inhibits both processes. The effect of DTT on DCCD-inhibited FoF1-ATPase activity of R. sphaeroides membrane vesicles has been analyzed too. DTT increases the DCCD-inhibited ATPase activity. Thus, more negative values of Eh by addition of DTT might regulate FoF1-ATPase activity. The participation of the ATPase in redox sensing under photo-fermentative H2 production is suggested. This enzyme might be a target, having a significant role in these processes of purple bacteria.

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.

Photo-fermentative hydrogen production from mixed substrate by mixed bacteria

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

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products.

BackgroundBiological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium.ResultsAs continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control.ConclusionsWe have found that SMPs are the key factor in photo-fermentative H2 production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.

Effect of carbon and nitrogen sources on photo-fermentative H2 production associated with nitrogenase, uptake hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides KD131

During photo-fermentative H2 production, the effects of carbon and nitrogen sources on nitrogenase and hydrogenase activity, poly-β-hydroxybutyrate accumulation were investigated. In succinate/ammonium sulfate medium, H2 was not detected for the first 6h because high ammonium concentration considerably reduced the nitrogenase activity to below 5nmol/g-dcw/h. After 24h, 99% of the ammonium was consumed, and the nitrogenase activity increased to 296nmol/g-dcw/h, accelerating H2 production. In contrast, the ammonium in succinate/glutamate medium was much less, which led to rapid H2 production in the beginning. However, H2 evolution was repressed over time by increased ammonium. In the presence of H2, hydrogenase activity increased with time regardless of the nitrogen source, and consequently, H2 production was reduced. Compared with succinate, H2 production in acetate media was severely limited due to increased pH over 9. During extended cultivation, the PHB accumulated in acetate media was 7 times higher than in succinate media.

Effect of substrate concentration on continuous Photo-fermentative hydrogen production from lactate using Rhodobacter sphaeroides

The information on continuous operation and the use of actual waste as a feedstock are essential for the practical application of photo-fermentative H2 production. For the first 200 days, continuous H2 production from lactate was attempted using purple non-sulfur (PNS) bacteria, Rhodobacter sphaeroides KD131, under an illumination of 110 W/m2. During the continuous operation, 30% of the fermenter volume was replaced by fresh feedstock once a day, and substrate concentration was gradually increased from 5 mM to 30 mM. H2 production was negligible at 5 mM, which was ascribed to the fact that the electrons contained in lactate were mostly consumed for cell growth and soluble microbial products (SMPs) production. As lactate concentration increased, H2 production gradually increased and reached a maximum at 20 mM, showing a substrate conversion efficiency (SCE) of 38%, a H2 yield of 2.3 mol H2/mol lactateadded, and a H2 production rate of 309 mL H2/L-fermenter/d. Further increases of lactate concentration resulted in a drop of H2 production (<1.0 mol H2/mol lactateadded). When the feedstock was changed to actual waste obtained from a 1-day lactate fermentation of food waste, stable H2 production was maintained, but showed a decreased SCE of 24%. It was speculated that the low performance was due to the fact that actual waste contained not only pure lactate but also other organic compounds that could not be utilized by PNS bacteria. In addition, compared to feeding with pure lactate, the electron consumption to the cell growth was higher in feeding with actual waste, which led to the lower performance.

Semi-continuous photo-fermentative H2 production by Rhodobacter sphaeroides: Effect of decanting volume ratio

In this study, a semi-continuous operation of photo-fermentative H2-producing reactor was attempted at various decanting volume ratios (DVR, decanting volume per day/total working volume, %), ranging 30–70%, using Rhodobacter sphaeroides KD131. H2 production was not efficient with showing low H2 yields of 0.2 and 0.5molH2/mol succinateadded at 30% and 40% DVR, respectively. The low performance ascribed to the fact that over 70% of substrate electrons were diverted towards cell growth under these conditions. Meanwhile, cell growth was limited at DVR⩾50%; therefore, higher H2 yields (>2.0molH2/mol succinateadded) were observed. Both the highest H2 yield of 3.7molH2/mol succinateadded and production rate of 1494mL H2/L-reactor/d were achieved at 60% DVR. The content of soluble microbial products (SMPs) was measured, which accounted for 3–15% of substrate electrons. It was found that the largest (65–75%) portion of SMPs comprised low molecular-weight (<3kDa).

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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.