Effects of nutrient deprivation on biochemical compositions and photo-hydrogen production of Tetraselmis subcordiformis

Tetraselmis subcordiformis (formerly called Platymonas subcordiformis), a marine green alga, was previously demonstrated to photo-biologically produce hydrogen when treated with CCCP (Carbonyl Cyanide m-Chlorophenylhydrazone). The current results of our studies showed that there was a peak yield of hydrogen by T. subcordiformis at the stationary stage of its algal growth curve, suggesting that starvation of an essential element induced biochemical changes that enhanced hydrogen production by T. subcordiformis. Further investigation indicated that among nitrogen, sulphur and phosphorus (which are major components in the cultivation medium), nitrogen deprivation in the medium reduced the protein content inside the cells of T. subcordiformis, but increased the carbohydrate content over 4 times, resulting in 5.5 times increase in the hydrogen yield.     

Preparation of a microalgal photoanode for hydrogen production by photo-bioelectrochemical water-splitting

In this study, a microalga Tetraselmis subcordiformis (synonym: Platymonas subcordiformis)-based photoanode was prepared by a novel method developed in our lab. The optimal photocurrent density of microalgae photoanode, 37 μA/cm², was achieved under illumination of 145 μmol s⁻¹ m⁻² at anode potential of 0.5 V vs Ag|AgCl|sat. KCl, immobilized cell density of 2.08 × 10⁶/cm² and BQ concentration of 300 μmol/L. The results of measurements showed that oxygen evolution peak, hydrogen evolution peak and photocurrent response were all synchronous to light impulse in a three-electrode system. It revealed that there occurred a process of photo-bioelectrochemical water-splitting. Hydrogen can be produced by the method. The investigation for whole photo-bioelectrochemical process also indicated that the electrons for hydrogen evolution had two sources, microalgal metabolic process in dark condition and photosynthetic water oxidation. The photo-hydrogen evolution was twice more than hydrogen evolution in dark condition.     

Regulation of hydrogen production by Enterobacter aerogenes by external NADH and NAD

Experiments involving the addition of external nicotinamide adenine dinucleotide, reduced form (NADH) or nicotinamide adenine dinucleotide (NAD⁺) have been designed to examine how the hydrogen in Enterobacter aerogenes is liberated by NADH or NAD⁺. The addition of external NADH or NAD⁺ was found to regulate hydrogen production by E. aerogenes in resting cells, batch cultures, and chemostat cultures. Particularly in chemostat cultivation, with the external addition of NADH, hydrogen production via the NADH pathway was decreased, while that via the formate pathway was increased; in the end, the overall hydrogen p was decreased. The addition of NAD⁺, on the other hand, gave the opposite results. The membrane-bound hydrogenase was found to play a central role in regulating hydrogen production. The occurrence of NADH oxidation (NAD⁺ reduction) on the cell membrane resulted in an electron flow across the membrane; this changed the oxidation state and metabolic pattern of the cells, which eventually affected the hydrogen evolution.     

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.     

Study of hydrogen production by three strains of Chlorella isolated from the soil in the Algerian Sahara

In this study, the photosynthetic hydrogen production rates by some strains of green microalgae were investigated. Three strains of Chlorella isolated from arid soil and foggaras's water in the Algerian Sahara were used. Chlorella sorokiniana strain Ce, Chlorella salina strain Mt and Chlorella sp strain Pt6 produced hydrogen gas under sulphur-deprived conditions, but its rate was dependent on strain type and oxygen partial pressure in medium. In C. sorokiniana strain Ce, the maximum value of hydrogen accumulated was 147 ml at 222 h at 2% of O₂ pressure. Compared to C. sorokiniana strain Ce, C. salina strain Mt and Chlorella sp strain Pt6 produced less amount of hydrogen, but they were able to sustain with an O₂ partial pressure of up to 11–15.4%. Our data were compared with hydrogen production by Chlamydomonas reinhardtii. In this communication, the relationship between physiological behaviour, biochemical characteristic (starch and protein) and rates gas production (O₂ and H₂) was also specified.     

Potassium deficiency,a “smart” cellular switch for sustained high yield hydrogen production by the green alga Scenedesmus obliquus

Hydrogen is considered to be the future optimal energy carrier, and is expected to contribute to the growth of the world's economy by facilitating a stable supply of energy. The ability of green algae to produce hydrogen was discovered 74 years ago. Since then, several attempts were made, to increase hydrogen production yields, sulfur starvation being the best known. The main concern during these attempts was that the achievable increase in yield was not sustainable. In this contribution, potassium deficiency is presented as a biochemical/bioenergetic switch for a sustained high yield of hydrogen production via the photosynthetic apparatus. Potassium can partially be replaced by sodium in the majority of biochemical processes and as a result the system remains functional. However, sodium cannot replace potassium in the conversion of glucose to starch. This fact significantly increased the yield of hydrogen production through the Photosystem II independent pathway, since electrons originating from the metabolism of glucose are used in the continuous donation to the plastoquinone-pool of the photosynthetic electron chain. Additionally, PSII inactivation (and therefore the inhibition of O₂-production), the further synthesis and over activation of Photosystem I and plastidic hydrogenase, generated a sustained increase in hydrogen production, mainly through the PSII-independent pathway. The self regulation of these multistage processes in hermitically closed static systems of Scenedesmus obliquus cultivation, permitted the establishment of anoxic conditions and the continuous electron supply to highly activated hydrogenase, resulting in the sustained high yield hydrogen production and paving the way for future usage in an industrial scale application.     

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.     

Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

A brief look at three decades of research on cyanobacterial hydrogen evolution

Hydrogen evolution by cyanobacteria is a potential way of biohydrogen production for the future. The basic and early applied research over the last 30 years has established the basis of present knowledge in the field and is a platform for future R&D directions. This work briefly surveys some of the progress made in the field of cyanobacterial hydrogen evolution during this time period.     

Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus

The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In the present study, the relationship between light intensity and hydrogen production has been modelled in order to predict both the rate of hydrogen production and the amount of hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using lactate as carbon and hydrogen source. The rate of hydrogen production increased with increasing light intensity in a manner that was described by a static Baly model, modified to include the square of the light intensity. In agreement with previous studies, the kinetics of substrate utilization and growth of R. capsulatus was represented by the classical Monod or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen.     

Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration

Escherichia coli produces molecular hydrogen (H₂) during glucose fermentation. This production of H₂ occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H₂ producing rate (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ by Hyd-4 (hyf) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621 (hyfB-R  ) mutants (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was decreased ∼6.7 and ∼5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was severely decreased ∼7.8 and ∼3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H₂ producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H₂ production by E. coli during fermentation.     

Metabolomics of photobiological hydrogen production induced by CCCP in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii can produce hydrogen gas (H₂) in the presence of the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The addition of 15 μM CCCP to the algal cultures led to 13-fold increase in H₂ photoproduction compared to the control cultures without CCCP treatment. CCCP completely inhibited the photochemical activity of photosystem (PS) II under illumination. In order to better understand metabolic conditions necessary for sustained H₂ production, we have used gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) for metabolomics analysis that is independent of nutritional stress, specifically, sulfur deprivation, which had been used previously to induce H₂ photoproduction. Even 10 min after addition of CCCP, metabolites from many metabolic modules were found drastically decreased, including levels of free amino acids, unsaturated free fatty acids and nucleotides. During prolonged CCCP exposure H₂ production was found to be stable for at least 12 h with a continued increase in levels of free fatty acids. These results indicate that CCCP might become a useful treatment for production of biohydrogen in reactors. The increase in fatty acid production might then be a useful addition for production of carbon-derived biofuels.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Genome based metabolic flux analysis of Ethanoligenens harbinense for enhanced hydrogen production

Ethanoligenens harbinense is a promising hydrogen producing microorganism due to its high inherent hydrogen production rate. Even though the effect of media optimization and inhibitory metabolites has been studied in order to improve the hydrogen productivity of these cultures, the identification of the underlying causes of the observed changes in productivity has not been targeted to date. In this work we present a genome based metabolic flux analysis (MFA) framework, for the comprehensive study of E. harbinense in culture, and the effect of inhibitory metabolites and media composition on its metabolic state. A metabolic model was constructed for E. harbinense based on its annotated genome sequence and proteomic evidence. This model was employed to perform MFA and obtain the intracellular flux distribution under different culture conditions. These results allow us to identify key elements in the metabolism that can be associated to the observed production phenotypes, and that can be potential targets for metabolic engineering in order to enhanced hydrogen production in E. harbinense.     

Improvement of hydrogen production of Chlamydomonas reinhardtii by co-cultivation with isolated bacteria

Three bacteria, named L2, L3 and L4, were isolated from contaminated cultures of Chlamydomonas reinhardtii strain cc849 in laboratory. The phylogenetic analysis based on 16S rDNA sequences showed that L2, L3 and L4 belonged to genus Stenotrophomonas, Microbacterium and Pseudomonas, respectively. The co-cultivation of isolated L2, L3 and L4 with purified algae, respectively, demonstrated that moderate bacterial concentration did not affect algal growth significantly but improved algal H₂ production obviously. The maximal H₂ yields were gained by the co-culture of algae with L2 or L4, about 4.0 times higher than that of the single algal culture. Increased respiration rate or O₂ consumption was the main reason for the enhancement of H₂ yield of the co-cultures.     

Bioproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents

A preliminary study on photoproduction of hydrogen by Rhodobacter capsulatus KU002 isolated from leather industry effluents under different cultural conditions with various carbon and nitrogen sources was investigated. Hydrogen production was measured using a Gas chromatograph. Lactate promoted more amounts of hydrogen production under anaerobic light conditions and aerobic light conditions. Cumulative hydrogen production by the organism was recorded at various time intervals. Incubation period of 120 h was optimum for production of hydrogen. pH 7.0 ± 0.2 was optimum for production of hydrogen by growing cells, while pH 7.5 ± 0.26 for resting cells. l-cystine was a good nitrogen source for production of hydrogen. Growing cells produced more amount of hydrogen than resting cells. Glutamine was a poor nitrogen source for hydrogen production by Rb. capsulatus. Significance of the above results in the presence of existing literature is discussed.     

Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV

Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate and sodium carbonate) and compound carbon sources (malate and succinate, lactate and succinate). The concentrations of sole carbon sources on hydrogen production were investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield was 424 mmol H₂/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H₂/mol-substrate for 2.02 g lactate and 2.0 g succinate as the compound carbon source. The results showed hydrogen production for the compound carbon source was better than the sole carbon source.