The Study Of Medium Chain Fatty Acid Prodcution Through Chain Elongation With Different Electron Donor

Chain elongation technology based on anaerobic fermentation system for medium chain fatty acids production is a promising biochemical production technology.Electron donor and acceptor are the basis of chain elongation.Among them,electron donors provide carbon source and energy for microorganisms,and their types and properties will directly affect the pathway of chain elongation,products type and yield.Some studies suggest that propanol is a potential electron donor,which may have different chain elongation pathways and mechanisms.According to the three-carbon properties of propanol,it is speculated that propanol may be oxidized into propionyl coenzyme A and then enter the carbon chain lengthening cycle,which may realize the odd chain elongation and the selective production of odd carbon fatty acids.Due to the lack of relevant research on propanol,the metabolic pathway and mechanism of propanol have not been explained yet.Therefore,this paper is devoted to explore the characteristics and mechanism of propanol chain elongation.Firstly,the feasibility of propanol chain elongation and the appropriate reaction conditions were determined by comparing the reaction parameters like different electron acceptors,electron acceptor load,pH and temperature.Secondly,ethanol was selected as the comparison object,and compare the reaction characteristics of ethanol and propanol when they bound to different electron receptors.The characteristics and mechanism of propanol chain elongation were revealed and verified through batch experiments.Finally,microbial diversity analysis elucidate the effects of different electron donors and electron acceptors on the microbial communit,identify the similarities and differences between different samples.Representative samples were selected for subsequent analysis.Metagenomic and metaproteomics analysis were used to construct electron donor oxidation and chain elongation pathways of propanol,reveal key enzymes and microorganisms.The main conclusions of this paper are as follows:（1）When the mixed anaerobic microbial community uses propanol as electron donor,it can only directly combine with acetate to generate medium chain fatty acid.Acetate is necessary for propanol chain elongation.When the temperature was 30 ℃ and the pH was 6,the production rate and selectivity of medium chain fatty acids were high when using propanol and acetate.Valerate and heptanoate were main product,with a small amount of butyrate and caproate.Propanol（100-300 mM）has a stronger inhibition effect than ethanol（200-400 mM）.Propanoate produced by propanol oxidation will inhibit the propanol oxidation and chain elongation.The inhibition can be alleviated by increasing acetate concentration.The optimal ratio of propanol/acetate（1:1）was lower than that of ethanol/acetate（3:1）,possibly because propanol-acetate had different chain elongation pathway and mechanism.It is preliminarily concluded that propanol needs to use acetate for even chain elongation.（2）The chain elongation products of both ethanol and propanol conform to the even number chain elongation,but propanol can only use acetate as electron acceptor.The metabolic mechanism of propanol-acetate was speculated.Propanol was oxidized to propionyl-CoA first,and then further oxidized to propionate.Hydrogen and energy（ATP）released from this process were used to reduce acetate to acetyl-CoA.After that,acetyl-CoA enters chain elongation cycle（for example,the reverse β-oxidation cycle）,and add two carbons to the electron acceptor at the end of each cycle.Odd chain fatty acid have stronger inhibitory effect than even chain fatty acid,which makes it difficult for microorganisms to synthesize and accumulate high concentration of odd chain fatty acid.（3）According to the results of microbial diversity analysis,the dominant bacteria genus in the reactor capable of chain elongation is Clostridium_sensu_stricto₁2 and Desulfovibrio.Clostridium_sen-su stricto₁2 is a genus that has been proven to have complete chain elongation function,while Desulfovibrio may be related to interspecific electron transfer and sulfonate degradation.Oscillibacter、Caproiciproducens and Colidextribacter may also contribute to the chain elongation process.Through combination analysis of metagenomic and metaproteomics analysis,the key microorganism and metabolic mechanism of propanol and acetate chain elongation were further revealed.C.kluyveri and C.bacterium are dominant species with high abundance and high expression activity,which can construct complete chain elongation cycle.C.kluyveri can efficiently synthesize organic carboxylate from ethanol,propanol and acetate.C.bacterium have stronger tolerance to odd chain fatty acid such as propionate and valerate.The metabolic pathway of propanol and acetate chain elongation was established.Due to the absence of propanal dehydrogenase,propanol is oxidized to propanal and then directly converted to propionate with aldehyde dehydrogenase.Some bacteria use the reducing hydrogen released from propanol oxidation to reduce acetate to acetyl-CoA.Instead of producing methane,some methanogens convert acetate to acetyl adenylate and continue to convert it to acetyl-CoA.Eventually,acetyl-CoA is used as an electron donor and take part into chain elongation.A complete reverse β oxidation cycle and fatty acid synthesis pathway can be constructed in EA,EP and PA reactors.The relative richness of the enzymes related to the reverse β-oxidation cycle and the related bacterial species were higher,indicating that the reverseβ-oxidation cycle mainly occurred chain elongation pathway.