The activity of the Synechocystis PCC6803 AbrB2 regulator of hydrogen production can be post-translationally controlled through glutathionylation

We show that the Synechocystis AbrB2 repressor of hydrogen production, down regulates the defence against oxidative stress. The single widely conserved cysteine of AbrB2 is also shown to play a crucial role in AbrB2 oligomerisation, and in AbrB2-mediated repression of the hydrogenase encoding operon (hoxEFUYH) and a wealth of other genes. Very interestingly, our results indicate that this cysteine is the target of glutathionylation, which affects the binding of AbrB2 on the hox operon-promoter DNA, as well as the stability of AbrB2 at the non-standard temperature of 39 °C. Similarly, we show that the cysteine of the other hoxEFUYH regulator AbrB1 can also be glutathionylated in vitro. These novel findings will certainly stimulate the in depth analysis of the influence of glutathionylation on the production of hydrogen, a field totally overlooked so far. They also emphasize on the evolutionary conservation of glutathionylation, a process mostly described in eukaryotes, so far.     

Improved oxygen tolerance of the Synechocystis sp. PCC 6803 bidirectional hydrogenase by site-directed mutagenesis of putative residues of the gas diffusion channel

Although of potential biotechnological interest, photobiological H₂ production from microalgae and cyanobacteria is strongly limited due to the oxygen sensitivity of hydrogenases, the H₂-evolving enzymes. We study here the [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 to identify structural determinants of its sensitivity to O₂. Based on previous work on the hydrogenase from Desulfovibrio fructosovorans and on a structural model of the Synechocystis hydrogenase, we have created various mutants of the Synechocystis enzyme. Amino acids residues homologous to those defining the end of the intramolecular gas channel in the D. fructosovorans enzyme were specifically targeted, these residues being previously described as critical for enzyme activity and tolerance to O₂. We show here that mutation I64M of the Synechocystis enzyme alters gas diffusion kinetics and improves O₂ tolerance. These results constitute the first report demonstrating that an O₂ tolerance-related character could be transposed from a proteobacterial hydrogenase to a cyanobacterial one, and may constitute the first published improvement of O₂ tolerance of a cyanobacterial enzyme by single site-directed mutagenesis.     

Dark hydrogen production in nitrogen atmosphere – An approach for sustainability by marine cyanobacterium Leptolyngbya valderiana BDU 20041

Biological hydrogen production is an ideal system for three main reasons i) forms a renewable energy source, ii) gives clean fuel and iii) serves as a good supplement to oil reserves. The major challenges faced in biological hydrogen production are the presence of uptake hydrogenase and lack of sustainability in the cyanobacterial hydrogen production system. Three different marine cyanobacterial species viz. Leptolyngbya valderiana BDU 20041, Dichothrix baueriana BDU 40481 and Nostoc calcicola BDU 40302 were studied for their potential use in hydrogen production. Among these, L. valderiana BDU 20041, was found to produce hydrogen even in 100% nitrogen atmosphere which was 85% of the hydrogen produced in argon atmosphere. This is the first report of such a high rate of production of hydrogen in a nitrogen atmosphere by a cyanobacterium, which makes it possible to develop sustained hydrogen production systems. L. valderiana BDU 20041, a dark hydrogen producer uses the reductant essentially supplied by the respiratory pathway for hydrogen production. Using inhibitors, this organism was found to produce hydrogen due to the activities of both nitrogenase and bidirectional hydrogenase, while it had no ‘uptake’ hydrogenase activity. The other two organisms though had low levels of bidirectional hydrogenase, possessed considerable ‘uptake’ hydrogenase activity and hence could not release much hydrogen either in argon or nitrogen atmosphere.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation

The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.     

An in silico re-design of the metabolism in Thermotoga maritima for increased biohydrogen production

Microbial hydrogen production is currently hampered by lack of efficiency. We examine how hydrogen production in the hyperthermophilic bacterium Thermotoga maritima can be increased in silico. An updated genome-scale metabolic model of T. maritima was used to i) describe in detail the H₂ metabolism in this bacterium, ii) identify suitable carbon sources for enhancing H₂ production, and iii) to design knockout strains, which increased the in silico hydrogen production up to 20%. A novel synthetic oxidative module was further designed, which connects the cellular NADPH and ferredoxin pools by inserting into the model a NADPH-ferredoxin reductase. We then combined this in silico knock-in strain with a knockout strain design, resulting in an in silico production strain with a predicted 125% increase in hydrogen yield. The in silico strains designs presented here may serve as blueprints for future metabolic engineering efforts of T. maritima.     

Hydrogen production by Escherichia coli without nitrogen sparging and subsequent use of the waste culture for fast mass scale one-pot green synthesis of silver nanoparticles

In view of the transition to hydrogen as a major energy carrier in the future, new routes for bringing down the cost of biological hydrogen production need to be explored. The current study was devoted to optimizing the dark fermentation by Escherichia coli HD701 for hydrogen production from an acid-hydrolyzed potato starch residue stream without nitrogen sparging to reduce the cost. To further increase the economic feasibility of hydrogen production by E. coli, this study explores the use of the waste culture after hydrogen production in mass scale one-pot green synthesis of silver nanoparticles.     

Enhancement of hydrogen production by the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005

The efficiency of hydrogen production by different cyanobacterial species depends on several external factors. We report here the factors enhancing hydrogen production by filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Cells adapted to dark-anaerobic conditions produced hydrogen consistent with increased hydrogenase activity when supplemented with Fe²⁺. Stimulation of hydrogen production could be achieved by addition of reductants, either dithiothreitol or β-mercaptoethanol with higher production observed with the latter. Additionally, Fe²⁺ and β-mercaptoethanol added to nitrogen- and sulphur-deprived cells significantly stimulated H₂ production with maximal value of 5.91 ± 0.14 μmol H₂ mg Chla⁻¹ h⁻¹. Glucose and a small increase of osmolality imposed by either NaCl or sorbitol enhanced hydrogen production. High rates of hydrogen production were obtained in cells adapted in nitrogen-deprived medium with neutral and alkaline external pH, significant decrease of hydrogen production occurred under acidic external pH.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

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.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Expression of aldehyde dehydrogenase gene increases hydrogen production from low concentration of acetate by Rhodobacter sphaeroides

Rhodobacter sphaeroides RV (RV) is a hydrogen-producing bacterium exhibiting the highest yield of hydrogen production from organic acids such as lactate and acetate, which are the byproducts of hydrogen fermentation by hydrogen-producing anaerobic bacteria. Co-fermentation of the RV strain with anaerobic bacteria is an efficient method of hydrogen production. However, less than 21 mM acetate is produced by the anaerobic bacteria, which is too low for efficient hydrogen production by the RV strain; it requires approximately 75 mM acetate. In this study, 2 distinct isozymes of aldehyde dehydrogenase from Rhodospirillum rubrum were separately overexpressed in the RV strain. The recombinant RV strains that were designated as RVAD1 and RVAD2, exhibited 13-fold higher ALDH activities than the wild-type RV strain. Hydrogen yields of both of the recombinant strains were 1.4-fold higher than that of the RV strain in 21 mM acetate. In 43 mM acetate, the RVAD1 strain showed higher yield, though the RVAD2 strain showed lower yield as compared to that of the RV strain. In 64 mM acetate and all concentrations of lactate tested (21, 43 and 64 mM), the yields of the recombinant strains were lower than those of the RV strain. The intact (empty) expression plasmid increased the ALDH activity and had little effect on the hydrogen production in acetate, however, it decreased the production in lactate. At the beginning of the fermentation process, when very little hydrogen had been produced, the recombinant strains expressing the ALDH gene consumed smaller amounts of acetate compared to the wild-type strain. We have discussed the effects of ALDH on hydrogen production in this report.     

The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations

This study evaluates the effect of pH (4-7) on fermentative biohydrogen production by utilizing three isolated Clostridium species. Fermentative batch experiments show that the maximum hydrogen yield for Clostridium butyricum CGS2 (1.77 mmol/mmol glucose) is achieved at pH 6, whereas a high hydrogen production with Clostridium beijerinckii L9 (1.72 mmol/mmol glucose) and Clostridium tyrobutyricum FYa102 (1.83 mmol/mmol glucose) could be achieved under uncontrolled pH conditions (initial pH of 6.4-6.6 and final pH of 4-4.2). Low hydrogen yields (0-0.6 mmol/mmol glucose) observed at pH 4 are due likely to inhibitory effects on the microbial growth, although a low pH can be thermodynamically favorable for hydrogen production. The low hydrogen yields (0.12-0.64 mmol/mmol glucose) observed at pH 7 are attributed not only to thermodynamically unfavorable, but also metabolically unfavorable for hydrogen production. The relatively high levels of lactate, propionate, or formate observed at pH 7 reflect presumably the high enzymatic activities responsible for their production, together with the low hydrogenase activity, resulting in a low hydrogen production. A correlation analysis of the data from present and previous studies on biohydrogen production with pure Clostridium cultures and mixed microflora indicates a close relation between the hydrogen yield (YH₂) and the (YH₂)/(2(YHAc+YHBu)) ratio, with the observed correlation coefficient (0.787) higher than that (0.175) between YH₂ and the molar ratio of butyrate to acetate (B/A). Based on the (YH₂)/(2(YHAc+YHBu)) ratios observed at different pHs, a control of pH at 5.5-6.8 would seem to be an effective means to enhance the fermentative biohydrogen production.     

Influence of Escherichia coli hydrogenases on hydrogen fermentation from glycerol

Since the actual role of Escherichia coli hydrogenases on fermentation from glycerol has not been clear, we evaluated the effect of inactivation of each E. coli hydrogenase on cell growth, hydrogen production, organic acids production, and ethanol production. Inactivation of hydrogenase 2 and hydrogenase 3 reduced cell growth, hydrogen and succinate production as well as glycerol utilization while acetate increased. Inactivation of hydrogenase 2 in minimal medium at pH 7.5 impaired hydrogen production, but no significant effect occurred at pH 6.5 or in complex medium. Inactivation of hydrogenase 3 impaired hydrogen production in minimal and rich medium, pH 6.5 and pH 7.5 accumulating formate in all conditions. Therefore during fermentation from glycerol, hydrogenase 3 is the main hydrogenase with hydrogen synthesis activity through the formate hydrogen lyase complex. Hydrogenase 2 seems mainly required for optimum glycerol metabolism rather than hydrogen synthesis. There were no significant impacts by inactivating hydrogenase 1 and hydrogenase 4.