Construction And Optimization Of Co-production System Of Hydrogen And Butyl Butyrate By Mixed Bacteria Fermentation

At present,the energy crisis and climate problems are becoming more and more serious.It is becoming more and more urgent for countries around the world to realize the vision of carbon neutrality and carbon peak,and to develop biological green hydrogen using renewable biomass as raw material.Dark fermentation hydrogen production technology has high hydrogen production,simple operation,strong industrial development potential,and techno-economic.However,the inhibitory effect of volatile fatty acids（VFAs）accumulated in the dark fermentation process on hydrogen accumulation has always been a difficult problem.To solve the inhibitory effect of VFAs on the hydrogen production process and further convert VFAs into high value-added esters,a mixed fermentation system for stable co-production of hydrogen and butyl butyrate was constructed by coupling the dark fermentation hydrogen production and in-situ esterification process.The following studies were conducted:（1）Lipase-catalyzed synthesis of butyl butyrate in the organic solvent-water biphasic system:To solve the problem of difficult separation of butyric acid in the fermentation broth,in-situ separation and high-value conversion of butyric acid was carried out by in-situ esterification.Firstly,the effects of different influencing factors on the synthesis of butyl butyrate catalyzed by lipase in an organic solvent-water two-phase system were investigated.Dodecane was selected as the extractant and lipase Eversa（?）Transform 2.0 was selected as the catalyst for the esterification reaction.The alcohol acid mixture of 3 g/L butanol and 2 g/L butyric acid was used as the substrate,the ratio of dodecane to the alcoholic acid mixed solution was 1:4,the working temperature was 35°C,the reaction p H value was 4.0,and the addition of lipase was 0.5%.The content of butyl butyrate in dodecane reached 2.11 g/L.The lipase Eversa（?）Transform 2.0 is beneficial to the esterification reaction to move toward the synthesis of butyl butyrate at a high molar ratio of butanol to butyric acid.（2）Investigation of the influencing factors of the mixed bacterial fermentation co-production of hydrogen and butyl butyrate system:Because of the mismatch between the consumption rate and the production rate of butyric acid in the co-production system,the influencing factors of co-production of hydrogen and butyl butyrate by mixed bacteria fermentation were explored,including the amount of lipase added,the concentration of substrate and the amount of butanol added.Finally,the optimal process parameters of the co-production system were determined.With the substrate concentration of 24 g/L starch,the ratio of extractant to fermentation broth was 1:4,and enzyme addition of 0.5%.Butyl butyrate in dodecane reached 8.66 g/L,hydrogen accumulation of 3631 m L/L,which was 58.17%higher than that of the control group,and the hydrogen yield was 1.23 mol H₂/mol glucose,and butanol supplementation was continued at the end of the hydrogen production fermentation.The accumulation of butyl butyrate in dodecane was further increased to 24.38 g/L.（3）Process optimization of co-production of hydrogen and butyl butyrate by mixed fermentation:To improve the yield of hydrogen and butyl butyrate,the fermentation process of the co-production system was further optimized.With 3 g/L butanol concentration as the feeding amount,butanol feeding was carried out in batches for many times.The hydrogen accumulation of the co-production system was not significantly inhibited,and the accumulation of butyl butyrate in dodecane reached 22.40 g/L.Co-production fermentation with biochar bacterial agents resulted in an accumulation of 3524 m L/L of hydrogen and 25.99 g/L of butyric acid butyl ester in dodecane.The addition of calcium carbonate can significantly shorten the fermentation time,the hydrogen accumulation reached 3389 m L/L,and the acidification degree of the fermentation broth was significantly reduced,so that the p H of the fermentation broth reached more than 5.6,which was not suitable for the esterification reaction of lipase Eversa（?）Transform 2.0,and the butyl butyrate in dodecane only reached 3.64 g/L.The substrate concentration was increased to 48 g/L starch,and calcium carbonate was added.The hydrogen accumulation in the co-production system reached 10793 m L/L.Compared with the batch fermentation of low substrate concentration of 24 g/L starch at the same concentration of 10g/L calcium carbonate,the cumulative amount of hydrogen in the co-production system increased by 211.78%,the hydrogen yield was 1.73 mol H₂/mol glucose,the cumulative amount of butyl butyrate in dodecane reached 21.08 g/L,and the ester yield was 0.10 g/g glucose.In summary,to solve the inhibition of the hydrogen production process by the accumulation of butyric acid in the fermentation broth,this paper constructed a system for the co-production of hydrogen and butyric acid butyl ester by mixed bacterial fermentation,which coupled the dark fermentation hydrogen production process with the in situ esterification process.On the one hand,the butyric acid accumulated in the fermentation broth was converted into high-value-added butyl butyrate by the action of lipase,and on the other hand,the co-production system weakened the inhibition of hydrogen accumulation by butyric acid.The co-production system constructed in this paper provides technical and theoretical support for the synthesis of other short-chain fatty acid esters,and also provides ideas for the integrated refining process of dark fermentation hydrogen production and other VFAs resources.