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.     

Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H₂/mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations.     

Hydrogen production by the thermophilic eubacterium Thermotoga neapolitana from storage polysaccharides of the CO2-fixing diatom Thalassiosira weissflogii

Planktic diatoms are the largest primary producers in marine and freshwater habitats. Their dry biomass accumulates up to 50% of lipids and contains water-soluble β-1,3-glucans as major storage products. Because of the world-wide abundance of these photosynthetic protists, β-1,3-glucans may rival cellulose as the polysaccharide with the highest annual production on Earth. Here we show the feasibility of a simple and efficient process leading to bio-hydrogen by dark fermentation of microalgal biomass with the thermophilic bacterium Thermotoga neapolitana. Production of the biogas on minimum medium supplemented only with the extract of the diatom Thalassiosira weissflogii proved that algal biomass per se can serve as substrate for sustaining the biotechnological process with no requirement of any pretreatment and external integration of other nutrients. At the same time, lipids unused for the anaerobic production of the biogas, can be employed for production of bio-diesel, thus considerably increasing the economic potential of these renewable feedstocks.     

Hydrogen production from carrot pulp by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

Hydrogen was produced from carrot pulp hydrolysate, untreated carrot pulp and (mixtures of) glucose and fructose by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana in pH-controlled bioreactors. Carrot pulp hydrolysate was obtained after enzymatic hydrolysis of the polysaccharide fraction in carrot pulp. The main sugars in the hydrolysate were glucose, fructose, and sucrose.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

Capnophilic lactic fermentation and hydrogen synthesis by Thermotoga neapolitana: An unexpected deviation from the dark fermentation model

The heterotrophic bacterium Thermotoga neapolitana produces hydrogen by fermentation of organic substrates. The process is referred to as dark fermentation and is typically complemented by production of acetic acid. Here we show that synthesis of products derived by reductive metabolism of pyruvate, mainly lactic acid, occurs to the detriment of acetic acid fermentation when the cultures of the thermophilic bacterium are flushed by saturating level of CO₂. Sodium bicarbonate in a very narrow range of concentrations (∼14 mM) also causes the same metabolic shift. The capnophilic (CO₂-requiring) re-orientation of the fermentative process toward lactic acid does not affect hydrogen productivity thus challenging the currently accepted dark fermentation model that predicts reduction of this gas when glucose is converted into organic products different from acetate.     

Distinctive properties of high hydrogen producing extreme thermophiles, Caldicellulosiruptor saccharolyticus and Thermotoga elfii

Growth and hydrogen production by two extreme thermophiles during sugar fermentation was investigated. In cultures of Caldicellulosiruptor saccharolyticus grown on sucrose and Thermotoga elfii grown on glucose stoichiometries of 3.3 mol of hydrogen and 2 mol of acetate per mol C₆-sugar unit were obtained. The hydrogen level was about 83% of the theoretical maximum. C. saccharolyticus and T. elfii reached maximum cell densities of 1.1×10⁹ and 0.8×10⁹ cells/ml, respectively, while their maximum hydrogen production rates were 11.7 and 5.1 mmol/g dry weight/h, respectively. For growth of C. saccharolyticus on sucrose, a biomass yield of 45.1 gDW/mol sucrose and a Y_ATP of 11.3–14.1 were calculated. Replacement of yeast extract by casamino acids, plus proline and vitamins in the medium of C. saccharolyticus resulted in similar yields of hydrogen production on sucrose, but diminished the rate by about 30%. Both yeast extract and tryptone were required by T. elfii, and appeared to function as sources of carbon, nitrogen and energy. In the absence of tryptone, T. elfii converted 26% of the glucose to another by-product, resulting in a lower yield of hydrogen. Growth of T. elfii ceased prior to glucose depletion, but the culture continued to ferment glucose to hydrogen and acetate until all glucose was consumed.     

Biohydrogen production from untreated and hydrolyzed potato steam peels by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana

Production of hydrogen by the extreme thermophiles Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana was studied in serum flasks and in pH-controlled bioreactors with glucose, and hydrolyzed and untreated potato steam peels (PSP) as carbon sources. Two types of PSP hydrolysates were used: one in which the starch in the PSP was liquefied with alpha-amylase, and one in which the liquefied starch was further hydrolyzed to glucose by amyloglucosidase.     

The proton iron-sulfur cluster environment of the [FeFe]-hydrogenase maturation protein HydF from Thermotoga neapolitana

[FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H₂. Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx₄₆-₅₃HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF from Thermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.     

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 co-cultures of Lactobacillus and a photosynthetic bacterium, Rhodobacter sphaeroides RV

Hydrogen production with glucose by using co-immobilized cultures of a lactic acid bacterium, Lactobacillus delbrueckii NBRC13953, and a photosynthetic bacterium, Rhodobacter sphaeroides RV, in agar gels was studied. Glucose was converted to hydrogen gas in a yield of 7.1 mol of hydrogen per mole of glucose at a maximum under illuminated conditions.     

Effect of temperature and iron concentration on the growth and hydrogen production of mixed bacteria

Anaerobic mixed culture acclimated with sucrose was used as inoculum in batch experiments to investigate the effects of various parameters on biological hydrogen production from sucrose. In particular, the effect of the culture temperature has been investigated in detail. The optimum of the iron concentration in the external environment on hydrogen production was also studied at different temperatures. Experimental results show that the hydrogen production ability of the anaerobic bacteria was deeply affected by both culture temperature and iron concentration. Increasing the culture temperature favored the production of hydrogen when it was in the range of 25–40 °C, and high sucrose conversion efficiencies (ca. 98%) were consistently obtained with the mixed bacteria at the same time. While the temperature went on increasing to 45 °C, the hydrogen production was almost inhibited. The optimum concentrations of iron for hydrogen production decreased obviously along with increasing the reactor's temperature. For 25, 35, and 40 °C, the maximum production yield of hydrogen were 356.0, 371.7, and 351.1 ml obtained at the iron concentration of 800, 200, and 25 mg FeSO₄l^-1${\mathrm{l}}^{-1}$, respectively.     

Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains

This study explored the genetic engineering of Escherichia coli for hydrogen (H₂) production. In E. coli W3110, the introduction of NAD⁺-reducing [NiFe]-hydrogenase from Cupriavidus necator, combined with the inactivation of three endogenous [NiFe]-hydrogenases, exhibited not only H₂ production but also H₂ uptake based on exogenous hydrogenase. Although the H₂ production ability was much lower than the H₂ uptake ability, inactivation of the ethanol, lactate, and succinate production pathways resulted in a marked increase in H₂ production, demonstrating the bidirectional hydrogenase function in vivo depending on NADH/NAD⁺. Unexpectedly, H₂ production was completely repressed under conditions for high expression of exogenous hydrogenase. Furthermore, the introduction of the heterologous enzyme markedly repressed the endogenous H₂ production ability of E. coli W3110 but not the HST02. These in vivo behaviors largely correlated with in vitro hydrogenase activity suggested complicated interactions between the native and nonnative functional expression of [NiFe]-hydrogenases.     

Prospecting hydrogen production of Escherichia coli by metabolic network modeling

Genome-scale model was applied to analyze the anaerobic metabolism of Escherichia coli. Three different methods were used to find deletions affecting fermentative hydrogen production: flux balance analysis (FBA), algorithm for blocking competing pathways (ABCP), and manual selection. Based on these methods, 81 E. coli mutants possessing one gene deletion were selected and cultivated in batch experiments. Experimental results of H₂ and biomass production were compared against the results of FBA. Several gene deletions enhancing H₂ production were found. Correctness of gene essentiality predictions of FBA for the selected genes was 78% and 77% in glucose and galactose media, respectively. 33% of the mutations that were predicted by FBA to increase H₂ production had a positive effect in experiments. Batch cultivation is a simple and straightforward experimental way to screen improvements in H₂ production. However, the ability of FBA to predict the H₂ production rate cannot be evaluated by batch experiments. Metabolic network models provide a method for gaining broader understanding of the complicated metabolic system of a cell and can aid in prospecting suitable gene deletions for enhancing H₂ production.     

Hydrogen production by Rhodopseudomonas palustris CQK 01 in a continuous photobioreactor with ultrasonic treatment

An ultrasonic treatment technique was applied to a continuous photobioreactor with Rhodopseudomonas palustris CQK 01 suspension to enhance photo-hydrogen production performance. After the start-up period, R. palustris CQK 01 suspension in the photobioreactor was intermittently agitated by ultrasonic wave with a frequency of 20 kHz and then the hydrogen production performance was evaluated. The ultrasonic agitation significantly dropped the hydrogen concentration in the suspension from 300 μmol/L to 50 μmol/L and thus increased the hydrogen production rate and hydrogen yield by nearly 2 times as compared with conventional photobioreactor. Furthermore, the effects of the ultrasonic power and time, influent substrate flow rate and concentration were investigated and discussed, respectively. The maximum hydrogen production performance of the continuous photobioreactor with ultrasonic treatment was obtained under the conditions of ultrasonic power 40 W, agitation/interval time 3/7 s, substrate concentration 75 mmol/L and substrate flow rate 40 ml/h, leading to the hydrogen production rate of 1.12 mmol/L/h and hydrogen yield of 0.23 mol-H₂/mol-glucose.     

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.     

Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs

Escherichia coli produces molecular hydrogen (H₂) during glucose or mixed carbon (glucose and glycerol) fermentation. Dependence of H₂ production rate (VH₂)$\left(V_{{\text{H}}_2}\right)$ on glucose at different pHs was studied in a concentration dependent manner. During growth of wild-type on glucose, increasing glucose concentration from 0.05% to 0.2% resulted in the marked inhibition of VH₂$V_{{\text{H}}_2}$. Inhibitory effect of glucose was shown at pH 7.5 and 6.5 but not pH 5.5. However, glycerol added in the growth medium with 0.1% glucose significantly increased VH₂$V_{{\text{H}}_2}$ but different effects at different pHs were established upon glucose or glycerol assays. The results indicate that H₂ production is inhibited by glucose in a concentration dependent manner during glucose fermentation but glucose in combination with glycerol might enhance H₂ production during mixed carbon fermentation.