Combined Hydrogen Production By Dark-and Photo-fermentation And Its Process Regulation

In order to replace fossil fuels and reduce air pollution,it is urgent to develop sources of renewable and clean energy.Bio-hydrogen is the most potential energy carrier for carbon neutralization and can provide sustainable energy supply.Compared with other biological fermentation systems,dark-and photo-fermentation hydrogen production can realize the efficient conversion of substrate,which is a favorable and practical choice to improve the efficiency of hydrogen production and expand the scale of hydrogen production technology.However,at present,due to the great difference in growth and metabolism between photo-fermentation bacteria and dark-fermentation bacteria,the system is very difficult to operate.In order to construct a reasonable dark-and photo-fermentation hydrogen production system,this subject carried out research from the aspects of screening and separating bacteria with high hydrogen production capacity,making a research on the growth properties of photosynthetic bacteria,optimizing the dark-and photo-fermentation hydrogen production process and exploring the regulation function of p H responsive transcription factor ThpacC,so as to achieve the purpose of high conversion efficiency of hydrogen production.（1）In order to enrich the germplasm resources of hydrogen producing microorganisms,a dark fermentation bacterium with high hydrogen production capacity was isolated from anaerobic activated sludge.It was identified as a new strain W-CB1 of Clostridium butyricum by molecular biological identification and phylogenetic analysis.Under the glucose concentration of 10 g/L,35℃water bath and magnetic stirring,the maximum hydrogen production of strain W-CB1 can reach 1.79 mol-H₂/mol-glucose,the average hydrogen content is about 50%,and the metabolic type is butyric acid fermentation.（2）In order to create an external environment suitable for the cooperative work of dark/photo fermentation bacteria,making a research on the growth properties of photosynthetic bacteria Rhodopseudomonas palustris DSM5859.Acetic acid,propionic acid,lactic acid,butyric acid and glucose can be used by R.palustris to grow,but they does not support the hydrogen production of R.palustris,and the macromolecular organic starch could not be used by R.palustris;The optimum conditions for the growth of photofermentative bacteria R.palustris were as follows:35℃,initial p H=7.0,light intensity 5.00 m W/cm²,and the ratio of sodium butyrate to sodium acetate 1:2.（3）Aiming at the problem of asynchronous growth and metabolism of dark light combined biological hydrogen production system,the effects of dark/photo fermentation bacteria ratio,L-cysteine concentration,Fe²⁺concentration and Ni²⁺concentration on combined hydrogen production were studied.Combined dark-and photo-fermentation had synergistic effect on hydrogen production,and the yield of H₂ was the highest when the ratio of C.butyricum to R.palustris was 1:3.When the concentration of L-cysteine was 4g/L,the cumulative hydrogen production and hydrogen concentration were the highest,and the hydrogen yield was increased to 2.20 mol-H₂/mol-glucose.The appropriate Fe²⁺concentration will increase the hydrogen production of the combined fermentation system.When the Fe²⁺concentration is 50 mg/L,the cumulative hydrogen production is 29.11%higher than that of the control group.Ni²⁺had no significant effect on hydrogen production by dark light combined fermentation.（4）In order to understand the p H related signal pathway and main regulatory genes of microorganisms,guide the design of stress resistant elements,and provide better adaptability for hydrogen producing bacteria under acid stress,the regulatory function of ThpacC was explored by culturing the knockout（KO-ThpacC）and overexpression mutant（OE-ThpacC）of the key regulator of p H response pathway in strain Trichoderma harzianum 3.9236under different initial p H and salt stress.Combined with the analysis of the changes of strain growth morphology,environmental p H and metabolites,it is found that ThpacC can adjust the environmental p H by affecting the concentration of metabolites,and there are other p H response pathways in acidic environment.In addition,ThpacC also plays a role in regulating T.harzianum to deal with salt stress.The lack of ThpacC may affect the balance of high osmotic potential and increase the sensitivity to salt stress.To sum up,starting with the screening of bacteria with high hydrogen production capacity,based on the growth and metabolism characteristics of photo-fermentation bacteria,this thesis constructed the dark-and photo-fermentation hydrogen production system,explored and optimized its process,and preliminarily explored the regulation function of p H response regulator ThpacC.This thesis can provide theoretical guidance and technical support for the construction and optimization of dark-and photo-fermentation hydrogen production system,provide reference for the design and construction of stress resistant elements,and is of great significance for accelerating the industrialization of biological hydrogen production technology.