Operation Optimization Of CSTR Hydrogen-Producing Reactor And Study On Metabonomics Of Hydrogen-Producing Bacteria

Dark fermentation anaerobic biological hydrogen production can effectively combine the treatment of high-concentration organic wastewater with hydrogen production.It has the advantages of wide sources of fermentation substrates,high conversion rate,strong hydrogen production capacity,and no need for light.It has become one of the research hotspots in the field of environmental protection and energy in recent years.Screening high-efficiency hydrogen-producing strains and carrying out metabonomic studies of hydrogen-producing bacteria are of great significance for understanding the hydrogen production mechanism of hydrogen-producing bacteria and improving hydrogen production efficiency.In this experiment,a continuous flow stirred tank reactor(CSTR)was used for hydrogen production by anaerobic fermentation.During the experiment,the influence of hydraulic retention time(HRT,12h,10h and 8h)and influent COD concentration(3000 mg·L^-1,5000 mg·L^-1 and 7000 mg·L^-1)on the operating efficiency of the reactor was investigated,discussed the COD removal rate,p H,effluent suspended solids(TS),activated sludge suspended solids mass concentration(MLSS)during the operation of the reactor and the change of the mass concentration of volatile suspended solids(MLVSS).Analyze the microbial flora structure and its evolution process in the activated sludge in the reactor with the help of high-throughput sequencing technology.A high-efficiency hydrogen-producing strain was screened from activated sludge using Hungate rolling tube technology,and then the abundance of different metabolites of this strain under different Fe²⁺concentration culture conditions was analyzed based on mass spectrometry and gas chromatography(GC-MS)technology Variety.The results show that when the influent COD concentration is controlled at about3000 mg·L^-1,the reactor works best when the HRT is 10 h.If the HRT is too long or too short,it will have a negative impact on the operation of the reactor.At this stage,the COD removal rate is about 30%,the p H is stable between 4.6?4.7,the cumulative hydrogen production is about 2 L·d^-1,and the total mass concentration of the final liquid phase fermentation product is 1089 mg?L^-1,mainly acetic acid,The formation of acetic acid type fermentation.The abundance of hydrogen-producing bacteria Ethanoligenens,Megasphaera,Enterobacter and Clostridium accounted for more than 40%of the total microorganisms in activated sludge.Continue to shorten the HRT to 8 hours,the COD removal rate fluctuates greatly and is unstable,and the cumulative hydrogen production decreases.The settling performance of activated sludge is reduced,and a large amount of sludge is lost with the effluent,resulting in the loss of activated sludge biomass.The abundance of hydrogen-producing bacteria in activated sludge dropped to about 16%.When the HRT is controlled for 10 hours and the influent COD concentration is about 5000 mg·L^-1,the maximum cumulative hydrogen production is obtained and the reactor is running well.Under this condition,the COD removal rate is about 26%,and the p H is stable between 4.5?4.6.The total mass concentration of liquid phase fermentation products was 2010 mg·L^-1,of which butyric acid content was the highest,1300 mg·L^-1,accounting for 51.5%of the total mass of liquid phase fermentation products,forming butyric acid type fermentation.The cumulative hydrogen production is about 2.5?3.0 L·d^-1.The abundance of hydrogen-producing bacteria Ethanoligenens and Megasphaera accounted for 18.57%and 13.81%,respectively,and the total abundance of hydrogen-producing bacteria accounted for more than 30%.When the influent COD concentration is 3000 mg·L^-1,the cumulative hydrogen production is low,and the operating efficiency of the reactor is poor.When the influent COD concentration is further increased to 7000 mg·L^-1,a large number of microorganisms will die due to the inability to adapt to the high concentration of organic substrates,causing the TS concentration to rise and the MLSS concentration to drop,and the cumulative hydrogen production from 2.5?3.0 L·d^-1 is reduced to 1.6?1.7 L·d^-1.The activated sludge was separated and screened by rolling tube,and a high-efficiency hydrogen-producing strain was selected as S-11.Using LM-1 as the basic medium,the cumulative hydrogen production after 30 hours of culture was 2230 m L/L-culture,and the specific production was The hydrogen rate is 0.89 mol H₂/mol glucose.The 16S r DNA identified this strain as a new strain of Enterobacter.The strain was optimized for culture with different Fe²⁺concentrations.When the Fe²⁺concentration was 0.2 g·L^-1,the highest specific hydrogen production rate was 1.09 mol H₂/mol glucose.When the Fe²⁺concentration is 0.5 g·L^-1,the specific hydrogen production rate is at least 0.61mol H₂/mol glucose.The Fe²⁺-free culture condition was selected as the control group,and the 0.2 g·L^{- 1}Fe²⁺and 0.5 g·L^-1Fe²⁺culture conditions were selected as the experimental group,and the metabolites were analyzed.A total of 83 different metabolites were identified between the 0.2 g·L^-1 Fe²⁺group and the Fe²⁺-free group,of which the abundance of 32metabolites was significantly up-regulated,and the abundance of 51 metabolites was significantly down-regulated.The metabolites with up-regulated abundance mainly include:riboflavin protein involved in electron transfer,plant alcohol,benzylamine,catechin,glutathione,4-aminomethylcyclohexanecarboxylic acid and other substances with reducing effect.It shows that Fe²⁺can reduce the oxidation-reduction potential in the fermentation environment,which is beneficial to the growth of anaerobic bacteria,thereby improving the hydrogen production capacity of Enterobacter S-11.A total of 68 different metabolites were identified between the 0.5 g·L^-1 Fe²⁺group and the Fe²⁺-free group,of which the abundance of 22 metabolites was significantly up-regulated,and the abundance of 46 metabolites was significantly down-regulated.The abundance of metabolites such as aniline,glycerol and phosphoethanolamine,which are involved in the breakdown of amino acids and lipids,increases,and the linoleic acid and other lipids involved in the synthesis of cell membranes decrease,indicating that excessive Fe²⁺concentration will bring Enterobacter S-11 Certain toxicity damages the cell membrane mechanism and weakens the effect of hydrogen production.