Sustained photobiological hydrogen production in the presence of N2 by nitrogenase mutants of the heterocyst-forming cyanobacterium Anabaena

Heterocyst-forming cells of the cyanobacterium Anabaena sp. strain PCC 7120 ΔHup, lacking an uptake hydrogenase, photobiologically produce H₂ by nitrogenase. Under N₂-rich atmosphere, the nitrogenase activity declines in a rather short time due to the sufficiency of combined nitrogen. From the parental ΔHup strain, site-directed double-crossover variants, dc-Q193S and dc-R284H, were created with amino acid substitutions presumed to be located in the vicinity of the FeMo-cofactor of nitrogenase. Unlike the case for the ΔHup strain, H₂ production activities of the variants were not decreased by the presence of high concentrations of N₂ and they continuously produced H₂ over 21 days with occasional headspace gas replacement. This property of N₂ insensitivity is a potentially useful strategy for reducing the cost of the culture gas in future practical applications of sustainable biofuel production. This Anabaena strain has only the Mo-containing nitrogenase which reduces acetylene to ethylene, but the dc-Q193S variant also produced ethane at low but measurable rates along with greater rates of ethylene production.     

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.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.     

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.     

Response of H2 production and Hox-hydrogenase activity to external factors in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

The effects of external factors on both H₂ production and bidirectional Hox-hydrogenase activity were examined in the non-N₂-fixing cyanobacterium Synechocystis PCC 6803. Exogenous glucose and increased osmolality both enhanced H₂ production with optimal production observed at 0.4% and 20 mosmol kg⁻¹, respectively. Anaerobic condition for 24 h induced significant higher H₂ase activity with cells in BG11₀ showing highest activities. Increasing the pH resulted in an increased Hox-hydrogenase activity with an optimum at pH 7.5. The Hox-hydrogenase activity gradually increased with increasing temperature from 30 ^○C to 60 ^○C with the highest activity observed at 70 ^○C. A low concentration at 100 μM of either DTT or β-mercaptoethanol resulted in a minor stimulation of H₂ production. β-Mercaptoethanol added to nitrogen- and sulfur-deprived cells stimulated H₂ production significantly. The highest Hox-hydrogenase activity was observed in cells in BG11₀-S-deprived condition and 750 μM β-mercaptoethanol measured at a temperature of 70 °C; 14.32 μmol H₂ mg chl a⁻¹ min⁻¹.     

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.     

Treatment with moderate concentrations of NaHSO3 enhances photobiological H2 production in the cyanobacterium Anabaena sp. strain PCC 7120

In cyanobacteria, treatment with low concentrations of NaHSO₃ can enhance photosynthetic efficiency, whereas NaHSO₃ in high amounts often inhibits cell growth and photosynthesis may even cause death. In the present study, our results showed that treatment with moderate concentrations of NaHSO₃ considerably improved the yield of photobiological H₂ production in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. Under steady state conditions, the accumulated H₂ levels in cells treated with 1 mM NaHSO₃ were approximately 10 times higher than that in untreated cells. Such improvement occurred in heterocysts and was most likely caused by increases in the expression and activity of nitrogenase. The effects of treatment with low, moderate, and high concentrations of NaHSO₃ in cyanobacteria were proposed on the basis of the results obtained in the present study and from previous knowledge.     

Relationship between molecular hydrogen production,proton transport and the F0F1-ATPase activity in Rhodobacter sphaeroides strains from mineral springs

This article aims to study hydrogen production and proton transport in two strains of purple non-sulfur bacterium Rhodobacter sphaeroides isolated from mineral springs of Armenia. This bacterium is able to grow and produce molecular hydrogen (H₂) in anaerobic conditions upon illumination. Along with H₂ production, a marked decrease in redox potential and the alkalization of the medium have been observed; the latter might be the evidence of proton influx. H₂ production and alkalization of the medium by whole cells both are suppressed by the F₀F₁-ATPase inhibitors – N,N′-dicyclohexylcarbodiimide (DCCD), sodium azide (NaN₃) and protonophore – carbonyl cyanide m-chlorophenylhydrazone (CCCP). Membrane vesicles of two strains of R. sphaeroides demonstrate ATPase activity, inhibited by DCCD and NaN₃, but not by CCCP. These results indicate a relationship between H₂ production, proton transport and the F₀F₁-ATPase activity that might be a pathway to regulate bacterial activity under anaerobic conditions.     

Enhanced hydrogen production by a newly described heterotrophic marine bacterium, Vibrio tritonius strain AM2, using seaweed as the feedstock

To achieve more stable bio-hydrogen (bioH₂) production from non-food feedstocks, stable feedstock preparations of marine biomass and an efficient bioH₂ system using marine bacteria under saline conditions are two important key technologies that needed to be developed. Vibrio tritonius strain AM2, which was isolated from the gut of a marine invertebrate, was cultured under various conditions in marine broth (at initial 2.25% (w/v) NaCl) supplemented with mannitol, a seaweed carbohydrate, to evaluate its hydrogen production. The maximum molar yield of bioH₂ was recorded as 1.7 mol H₂/mol mannitol at pH 6 and 37 °C. The mannitol-grown cells had higher yields of bioH₂ than the glucose-grown cells in the pH range 5.5–7.5. Compared to glucose, mannitol might be a better substrate for bioH₂ production using strain AM2. Fermentation product profiling revealed that strain AM2 might be utilising the formate-hydrogen pathway for bioH₂ production. Furthermore, strain AM2 was able to produce hydrogen from powdered brown macroalgae containing 31.1% dry weight of mannitol. The molar yield of hydrogen reached 1.6 mol H₂/mol mannitol contained in the seaweed feedstock. In conclusion, strain AM2 has the ability to produce hydrogen from mannitol with high yields even under saline conditions.     

Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18

Pantoea agglomerans BH18, isolated from mangrove sludge, could produce hydrogen under marine culture condition. To improve the hydrogen-producing capacity of this strain, we constructed a stable transposon-mutagenized library of P. agglomerans BH18. A Tn7-based transposon was randomly inserted into genomic DNA of P. agglomerans BH18. Mutants were identified by kanamycin resistance and amplification of the inserted transposon sequences. A transposon mutant, named as strain TB212, was screened for the highest hydrogen production ability. The total volume of hydrogen gas evolved by this mutant strain TB212 was 60% higher than that of the wild type. The mutant strain TB212 was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum initial pH of 7.0, and hydrogen production was 2.52 ± 0.02 mol H₂/mol glucose (mean ± S.E.) under marine culture condition. The mutant strain TB212 could produce hydrogen at the salt concentration from 3 to 6%. It was concluded that the transposon-mutagenized library may be a useful tool for investigation of high efficiency hydrogen-producing bacteria.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

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.