L-Cysteine Stimulated Hydrogen Production Process Of Hydrogen-Producing Bacteria

Fermentative bio-hydrogen production technology becomes one of the research focuses in environmental biotechnology, for its environmental friendly characters and producing clean energy. The fermentative bio-hydrogen production theory is improving, along with different genus hydrogen-producing bacteria are isolated, different forms reactor are developed, the culture condition are optimized and molecular biology are applied. The operation of demonstration project lays the foundation for the application of technology. Increasing the hydrogen production rate, reducing the cost and the stable running the hydrogen production reactors are essential to realize the industrialization of fermentative bio-hydrogen production.Researchers always try different methods to improve the hydrogen production rate. This research chose L-cysteine to increase the hydrogen production rate and analyse the mechanisms. On one hand, L-cysteine is susceptible to oxidization to give the disulfide derivative cystine, and the progress may low the oxidation-reduction potential. On the other hand, the side chain on cysteine is thiol, which serves an important structural role in many proteins. Thus, investigate how the L-cysteine effect the hydrogen-producing bacteria is significance to improve the hydrogen production rate, and may deepen the bio-hydrogen production theory and be used as reference for other methods improving the rate.In order to study how the L-cysteine worked on the hydrogen-producing bacteria, the pure culture and mixed culture were both reviewed, batch and continuous flow were both used and hydA was quantitative described by the real-time PCR. These methods help to analyze the effect theory exactly, and give an example for quantitative describe target gene from other hydrogen-producing bacteria.The ecological factors on growth and biogas production of the hydrogen-producing bacteria YUAN-3 were investigated from the perspective of the buffer solution. At the original pH of 6.0 in the buffer solution culture, YUAN-3 got maximum average specific hydrogen production rate of 5.75 mmolH₂/g-CDW·h and hydrogen yield of 2.32molH₂/mol-glucose. At the original pH of 7.0, YUAN-3 had most biomass of 0.59 g/L-culture.Batch culture of YUAN-3 showed L-cysteine improved the bacterial growth and hydrogen production and increase the substrate utilization capacity. Cell dry weight increased as added the L-cysteine concentration, when the dosage of L-cysteine was 1.5 g/L-culture, the biomass reached a maximum of 0.53 g/L-culture, the hydrogen production reached a maximum of 1995 mL/L- medium when the dosage of L-cysteine was 0.8 g/L-culture. The measured values and Matlab simulation of the modified Gompertz equation proved that adding L-cysteine accelerated the biogas production process of YUAN-3.The added of L-cysteine accelerated start-up of ethanol type fermentation, improved the hydrogen production rate and microbial activity, reduced the reactor ORP quickly. The reactor fed L-cysteine gradually formed a typical metabolites after 14d, and the reactor not fed L-cysteine had acetic acid and ethanol as the main metabolite of ethanol-type fermentation after 20d. At the 1st day, the ORP of the reactor fed L-cysteine immediately dropped to-400mv, and that needed more than 10d for the reactor not fed L-cysteine. The average hydrogen production rates were 2.74 L/d and 2.81 L/d for not fed and fed L-cysteine, respectively. The biomass were 9.5 L/d and 10.1 L/d for the two reactors at the end of the start-up.L-cysteine played a reductive role and the promoted the formation of the related enzymes in the hydrogen production of YUAN-3. The added of L-cysteine promoted the metabolic process of YUAN-3, improved the aggregation capacity and the extracellular polymer substance (EPS) had increased, too. The end liquid products ethanol and acetic acid played cumulative inhibition of the fermentation process, and low-pH led the fermentation to the termination.The added of L-cysteine reduced the activity of alcohol dehydrogenase in equal protein concentration, but because the biomass and unit of protein concentration in the biomass increased, the ethanol production also increased by added L-cysteine. Using hydA specific primer to describle the hydrogenase expression quantificationally, PCR results showed that L-cysteine concentration of 0.8 g/L- medium, the hydrogenase gene expression reached 2.72×10⁷ copies/μL. The increase of hydrogen production was because hydrogenase expression together with biomass increased.