Hydrogen Production with High Evolution Rate and High Yield by Immobilized Cells of Hydrogen-producing Bacteria Strain B49 in a Column Reactor

To improve the hydrogen evolution rate in continuous hydrogen production of a fermentative hydrogen-producing bacteria strain B49 (AF481148 in EMBL), 4% immobilized cells by polyvinyl alcohol-boric acid method, with the addition of a small amount of calcium alginate in a column reactor obtain hydrogen yield of 2.31 mol H2/mol glucose and hydrogen evolution rate of 1435.4 ml/L culture&middoth respectively at medium retention time of 2 h with a medium containing l0 g glucose/L. As the cell density in gel beads is increased to 8%, hydrogen yield and hydrogen evolution rate for l0 g glucose/L are 2.34 mol H2/mol glucose and 2912.4 ml/L culture &middot h respectively at medium retention time of 1 h, and for molasses wastewater COD of 7505.9 mg/L hydrogen production potential of 205.6 ml/g COD and hydrogen evolution rate of 2057.7 ml/L culture&middoth at hydraulic retention time of 0.75 h are observed. In the continuous culture pH value keeps around 3.9 by self regulation.

The start-up of biohydrogen-producing process by bioaugmentation in the EGSB reactor

Expanded granular sludge bed （EGSB） reactor and bioaugmentation were employed to investigate biohydrogen production with molasses wastewater. The start-up experiments consisted of two stages. In the first stage （0 - 24d） seeded with activated sludge, the butyric acid type-fermentation formed when the initial expanding rate, organic loading rate （OLR）, the initial redox potential （ORP） and hydraulic retention time （HRT） were 10%, 10.0 kg COD/（m^3·d）, -215 mV and 6.7 h, respectively. At the beginning of the second stage on day 25, the novel hydrogen-producing fermentative bacterial strain B49 （AF481148 in EMBL） were inoculated into the reactor under the condition of OLR 16. 0 kg COD/（m^3·d）, ORP and HRT about - 139 mV and 6.7 h, respectively, and then the reaction system transformed to ethanol-type fermentation gradually with the increase in OLR. When OLR, ORP and HRT were about 94.3 kg COD/（m^3·d）, -250 mV and 1.7 h, respectively, the system achieved the maximum hydrogen-producing rate of 282.6 mL H2/L reactor·h and hydrogen percentage of 51% -53% in the biogas.

Partial Characteristics of Hydrogen Production by Fermentative Hydrogen-producing Bacterial Strain B49

To investigate the characteristics of hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49 (AF481148 in EMBL), batch experiments are conducted under different conditions. Hydrogen production has a correlation with cell growth and the consumption of glucose and soluble protein. The optimum pH for cell growth is 4.5±0.15. At acidic pH 4.0±0.15, the bacteria has the maximum accumulated hydrogen volume of 2382 ml/L culture and the maximum hydrogen evolution rate of 339.9 ml/L culture·h with 1% glucose. The optimum temperature for cell growth and hydrogen production is 35℃. In addition, fermentative hydrogen-producing bacterial strain B49 can generate hydrogen from the decomposition of other organic substrates such as wheat, soybean, corn, and potato. Moreover, it can also produce hydrogen from molasses wastewater and brewage wastewater, and hydrogen yields are 137.9 ml H 2/g COD and 49.9 ml H 2/g COD, respectively.

Magnesium improves hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49

Batch experiments were conducted to investigate the effects of magnesium on glucose metabolism, including growth and hydrogen-producing capacity of fermentative hydrogen-producing bacterial strain B49. These abilities were enhanced with an increase in magnesium concentration. At the end of fermentation from （10 g/L） glucose, for 10 mg/L MgCl2·6H2O the cell growth in terms of optical density （OD） at 600nm was 0.46, the ratio of ethanol amount （mg/L） to acetate amount （mg/L） was 1.1, and the accumulated hydrogen volume was 934.9 mL H2/L culture; for 200 mg/L of MgCl2·6H2O OD600 nm was increased to 1.34. The accumulated hydrogen volume was increased to 2 360.5 mL H2/L culture, the ratio of ethanol amount （mg/L） to acetate amount （mg/L） was increased to 1.3 and polysaccharide was decreased to 2.5 mg/L. Moreover, the magnesium solution addition to the medium at different fermentation times affected hydrogen-producing ability. However, the later the addition time was postponed, the less the effect was on hydrogen evolution. Further experiments confirmed the enhancement was dependent on magnesium ions and not on the other inorganic ions such as SO42- or Cl-, which constituted the magnesium salts.

Length polymorphisms for intergenic spacer regions of 16S-23S rDNA in members of the new hydrogen-producing bacteria

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic spacer regions (ISR) was developed for the identification of species within the novel group hydrogen-producing anaerobes. The sizes of the PCR products varied from 1264 to 398 bp. Strain of isolate Rennanqilyf 3 was characterized as having products of 1262,398,638,437 and 436 bp. The isolate Rennanqilyf 1 had product of 1264 bp. The isolate Rennanqilyf 13 had products of 1261,579 and 485 bp. Of the 3 species of the novel group hydrogen-producing anaerobes examined, no one was indistinguishable. Two environmental isolates were identified as hydrogen-producing bacteria, which were new species in present taxon. Rennanqilyf 3 could not be associated with any Clostridium sp. studied. Rennanqilyf 1 could be classified into Clostridium genus. The combination between 16S rDNA equencing and length polymorphisms of IRS in 16S-23S rDNA is a better method for determining species of the hydrogen-producing bacteria.

A syntrophic propionate-oxidizing microflora and its bioaugmentation on anaerobic wastewater treatment for enhancing methane production and COD removal

Methane fermentation process can be restricted and even destroyed by the accumulation of propionate because it is the most difficult to be anaerobically oxidized among the volatile fatty acids produced by acetogenesis. To enhance anaerobic wastewater treatment process for methane production and COD removal, a syntrophic propionate-oxidizing microflora B83 was obtained from an anaerobic activated sludge by enrichment with propionate. The inoculation of microflora B83, with a 1：9 ratio of bacteria number to that of the activated sludge, could enhance the methane production from glucose by 2.5 times. With the same inoculation dosage of the microflora B83, COD removal in organic wastewater treatment process was improved from 75.6% to 86.6%, while the specific methane production by COD removal was increased by 2.7 times. Hydrogen-producing acetogene_sis.appeared to be a rate-limiting step in methane termentation, and the enhancement orhydrogen-producing acetogens in the anaerobic wastewater treatment process had improved not only the hydrogen-producing acetogenesis but also the acidogenesis and methanogenesis.