Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.     

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.     

Quantitative real-time PCR monitoring dynamics of Thermotoga neapolitana in synthetic co-culture for biohydrogen production

This study demonstrates the potential for biohydrogen production in a co-culture of two ecologically distant species, Thermatoga neapolitana and Caldicellulosiruptor saccharolyticus, and the development of a quantitative real-time PCR (qPCR) method for quantifying the hyperthermophilic bacterium of the genus Thermotoga. Substrate utilization and H₂ production performance was compared to those of their individual cultures. The highest H₂ yields obtained were 2.7 ± 0.05, 2.5 ± 0.07 and 2.8 ± 0.09 mol H₂/mol glucose for C. saccharolyticus, T. neapolitana, and their co-culture respectively. Statistical analysis comparing the H₂ production rate of the co-culture to either C. saccahrolyticus or T. neapolitana pure cultures indicated a significant difference in the H₂ production rate (p < 0.05: t-test), with the highest rate of H₂ production (36.02 mL L⁻¹ h⁻¹) observed from the co-culture fermentations. In order to monitor the presence of T. neapolitana in the bioprocess, we developed a qPCR method using 16S rRNA gene and hydrogenase (hydA) gene targets. The qPCR data using hydA primers specific to T. neapolitana showed an increase in hydA gene copies from 3.32 × 10⁷ to 4.4 × 10⁸ hydA gene copies per mL confirming the influence of T. neapolitana in the synthetic consortium.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Cloning and knockout of formate hydrogen lyase and H2-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A (△hycA), IAM1183-O (△hybO), and IAM1183-AO (△hycA/△hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 ± 38.59, 2747.203 ± 13.25 and 3372.019 ± 4.39 (ml h⁻¹ g⁻¹dry cell weight), respectively, higher than that of the wild strain (2321.861 ± 15.34 ml h⁻¹ g⁻¹dry cell weight). The total H₂ yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H₂/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H₂/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183△hycA/△hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L.     

Effect of iron concentration on continuous H2 production using membrane bioreactor

The effect of FeSO₄ on continuous H₂ production in a membrane bioreactor (MBR) was investigated using anaerobic mixed microflora under mesophilic condition. The H₂ production of 41.6 l/day was obtained at 10.9 mg FeSO₄/l, which was 1.59 times higher than that at 2.7 mg FeSO₄/l. Between 2.7 and 13.7 mg FeSO₄/l, the H₂ production rate increased in parallel with the H₂ yield under high-cell-density condition. For the same amounts of FeSO₄, increases in butyric acid together with decreases in lactic acid promoted a reduction of the number of protons and the resultant release of H₂. The hydrogenase activity of 1.08 mg methylene blue (M.B) reduced/min at 10.9 mg FeSO₄/l was about sixfold higher than at 2.7 mg FeSO₄/l. These results suggest that the addition of iron and sulfur to an MBR is an important key factor in the enhancement of H₂ production.     

Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum

The genes coding for two PII-like proteins, GlnB and GlnK, which play key roles in repressing the nitrogenase expression in the presence of ammonium ion, were interrupted from the chromosome of Rhodobacter sphaeroides. The glnB–glnK mutant exhibits the less ammonium ion-mediated repression for nitrogenase compared with its parental strain, which results in more H₂ accumulation by the mutant under the conditions. Rhodospirillum rubrum produces H₂ by both nitrogenase and hydrogenase. R. rubrum containing the recombinant pRK415 with an insert of hydC coding for its own Fe-only hydrogenase showed twofold higher accumulation of H₂ in the presence of pyruvate under photoheterotrophic conditions, which was not observed in the absence of pyruvate. The same was true with R. rubrum containing the recombinant pRK415 cloned with hydA coding for Fe-only hydrogenase of Clostridium acetobutylicum. Thus, Fe-only hydrogenase requires pyruvate as an electron donor for the production of H₂.     

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.     

Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii

The direct relationship between hydrogenase gene conformation and its function in green alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the gene region is present.     

SPEEK/epoxy resin composite membranes in situ polymerization for direct methanol fell cell usages

Sulfonated poly(ether ether ketone) (SPEEK)/4,4′-diglycidyl(3,3′,5,5′-tetramethylbiphenyl) epoxy resin (TMBP) composite membranes in situ polymerization were prepared for the purpose of improving the methanol resistance and mechanical properties of SPEEK membranes with high ion-exchange capacities (IEC) for the usage in the direct methanol fuel cells (DMFCs). The effects of introduction of TMBP content on the properties of the composite membranes were investigated in detail. The composite membranes have good mechanical, thermal properties, lower swelling ratio, lower water diffusion coefficient (0.87 × 10⁻⁵ cm² s⁻¹ at 80 °C) and better methanol resistance (5.26 × 10⁻⁷ cm² s⁻¹ at 25 °C) than SPEEK membranes. The methanol diffusion coefficients of the composite membranes are much lower than that of SPEEK membrane (17.5 × 10⁻⁷ cm² s⁻¹ at 25 °C). Higher selectivity was been found for the composite membranes in comparison with SPEEK. Therefore, the SPEEK/TMBP composite membranes show a good potential in DMFCs usages.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

Production of hydrogen from sewage biosolids in a continuously fed bioreactor: Effect of hydraulic retention time and sparging

Hydrogen was produced from primary sewage biosolids via mesophilic anaerobic fermentation in a continuously fed bioreactor. Prior to fermentation the sewage biosolids were heated to 70 °C for 1 h to inactivate methanogens and during fermentation a cellulose degrading enzyme was added to improve substrate availability. Hydraulic retention times (HRT) of 18, 24, 36 and 48 h were evaluated for the duration of hydrogen production. Without sparging a hydraulic retention time of 24 h resulted in the longest period of hydrogen production (3 days), during which a hydrogen yield of 21.9 L H₂ kg⁻¹ VS added to the bioreactor was achieved. Methods of preventing the decline of hydrogen production during continuous fermentation were evaluated. Of the techniques evaluated using nitrogen gas to sparge the bioreactor contents proved to be more effective than flushing just the headspace of the bioreactor. Sparging at 0.06 L L min⁻¹ successfully prevented a decline in hydrogen production and resulted in a yield of 27.0  L H₂ kg⁻¹ VS added, over a period of greater than 12 days or 12 HRT. The use of sparging also delayed the build up of acetic acid in the bioreactor, suggesting that it serves to inhibit homoacetogenesis and thus maintain hydrogen production.