Enhancement Of Caproate Production From Organic Waste Via Anaerobic Mixed Culture And Its Underlying Mechanisms

Caproate production via anaerobic mixed-culture is gaining widely interest.This is because the technology could upgrade low grade biomass to high value chemicals.In recent researches,this technology is faced with low productivity and high cost of electron donor.Essentially,these bottlenecks are originated in the unawareness of the mixed-culture caproate producing mechanisms and the regulating method.Thus,it is of critical importance to identify its key factor and the underlying regulation mechanisms to contribute to further application.This research firstly identified the key environment factors and the dominant species in mixed-culture caproate fermentation.Based on the findings of high E/A?ethanol to acid ratio?substrate requirement in caproate-producing phase,the ethanol-type acidification of organic waste was further studied to achieve authigenic electron donor production.Based on the inorganic carbon requirement in the caproate-producting phase,the regulation influences of inorganic carbon source addition on caproate production performance and microbial structure were studied.With these findings,this research used CHCl₃ as inhibitor to directionally constrain the competitive metabolic microbiome,and used CaCO₃ together with CHCl₃addition to enhance the caproate production,the caproate producing microbe was effectively enriched and caproate production performance was enhanced.In the last part,this research used anion exchange resin to alleviate the feedback inhibition of high concentration caproate.Thus,enhanced caproate production achieved and further caproate production enhancement mechanism was elucidated.With the assistant of next generation sequencing technology,qPCR,the microbial community structure and functional microbe dynamic under different performance were elucidated.By using RDA,Co-occurrence network analysis,the relation among key product,competitive and functional microbiome were studied and the mechanisms of enhancement was further elucidated.By using scanning electron microscope,the bacterial morphology in the microbiome was observed.The results indicated that:?1?The studies on identification of environment factors and key functional microbiome suggested that:In the acidification phase,inoculum to substrate ratio?ISR?dominated production profile.Under near-neutral and ISR 2:1 conditions,the organic waste could be effectively converted in carboxylate-type acidification.In caproate fermentation phase,ethanol to carboxylate ratio and pH are key parameters.Under the E/A 4:1,pH 7.5 optimal caproate production was reached at 15.0 g/L.While the caproate productivity and selectivity were 0.62 g/g and 88.2%,respectively.The next generation sequencing results indicated that caproate production in this system was dominated by Clostridium kluyveri,and the qPCR results indicated that caproate mainly produced as Clostridium kluyveri reached middle and late phase.In the late phase,caproate producing microbiome and caproate production decreased even when ethanol was supplied,and these production deterioration was correlated with the inorganic carbon shortage.?2?In order to gain authigene electron donor from organic waste,the ethanol-type acidification of organic waste was studied.The result suggested that organic waste could be converted in ethanol type acidification in natural acidification and yeast-inoculated condition,and the conversion characters in both condition were similar.The maximum ethanol production reached at 27.5 g/L and 31.9 g/L,which provided high E/A?5.7 and 10.4?fermentation broth and fitted the following caproate production requirement.Under sludge inoculated condition,the conversion efficiency was improved and the fermentation period was shorten,while the ethanol production was merely 6.9 g/L,the E/A was 0.24.The cellulase,hemicellulase,amylase,pectinase activity in groups with sludge inoculated were higher than those in groups under natural acidification and yeast inoculated condition.Studies on microbiome structure revealed that the bacterial community structure in natural acidification and yeast-inoculated condition were similar,their hydrolysis-functional microbiome were of rich diversity,while the ethanol-producing functional microbe were not the dominated species.Firmicutes and Proteobacteria contributed to 99%of relative abundance in both condition.However,the bacterial community structure in sludge inoculated groups was dramatically different from the other two group,Actinobacteria contributed to43.2%of all relative abundance.The fungal consortium studies showed that ethanol production was dominated by Saccharomycetaceae.Pichia membranaefaciens contributed to99.9%and 95.1%relative abundance in natrual acidification group and yeast-inoculated groups,respectively.While the inoculated species Saccharomyces cerevisiae was not the dominant species.?3?Studies on the regulation performance on caproate production via inorganic carbon addition revealed that inorganic carbon selection test indicated that CaCO₃ could sustain relatively stable inorganic carbon level in fermentation broth and could be suitable for caproate production enhancement.By increasing the CaCO₃ dosage,the ethanol ultilization rate increased from 1.27 to 2.32 g/?L·d?,the increment was 82.7%.However,caproate production was not increased,and side-effect including methane and butanol production were provoked.The qPCR results on key microbiome suggested that,Clostridium kluyveri growth was stimulated by increased CaCO₃ addition and the quantity was also maintained stable at relativelly high concentration,which were necessary to high caproate production.High-throughput sequencing results revealed that the relative abundance of caproate producting microbe Clostridium kluyveri increased as CaCO₃ addition increased,when 5,15,20,25 g/L CaCO₃ was added,the relative abundance of Clostridium kluyveri were 17.7%,18.0%,18.1%and 19.3%.Besides,the co-occurrence analysis indicated that homoacetogens and symtrophic were positive related to Clostridium kluyveri.?4?By introducing CHCl₃ in the caproate production,the inhibitory control performance of competitive metabolic pathway was studied.CHCl₃ as inhibitor could simultaneously and effectively suppress methanogen and homoacetogen within 0.5-5‰CHCl₃ concentration,methane and butanol production was restrained.CHCl₃ had slight side effect of on Clostridium kluyveri,meanwhile,in excessive CHCl₃ condition?10‰?,Clostridium kluyveri growth and caproate production could not sustain.Combining CHCl₃ and CaCO₃ addition,could achieve competitive pathway inhibition and production enhancement.Caproate production significantly?p<0.05?increased to 19.0-21.0 g/L.The scanning electron microscopy?SEM?on biomass showed that with the combination of CHCl₃ and CaCO₃,microbe in thick-form was more found.Next generation sequencing results suggested that the relative abundance of key functional microbe Clostridium kluyveri increased from 30.9-35.6%?with only CHCl₃?to 38.9-49.3%?the combination of CHCl₃ and CaCO₃?.The abundance of syntrophic bacterium was significantly reduced.Co-occurrence analysis also indicated that Bifidobactrerium pseudocatenulatum,which was capable to produce essential growth factor,co-related with Clostridium kluyveri.While species affiliated to Caproicproducen was independent to Clostridium kluyveri.?5?By using anion exchange resin to enhance caproate production,the studies suggested that in anion exchange resin selection test,SBAERs?strong base anion exchange resin?were of better performance than WBAERs.IRA-900 resin which was with 62 mg/g adsorption capacity,7.50 selectivity,and 88.2%elution efficiency,was better than other resins.During the desorption condition optimization studies and reusability test,IRA-900 desorption performance was not sensitive to temperature,by maintaining the mesophilic condition was enough.The concentration of NaOH solution as eluent reached at the optimal performance in1.5 mol/L,which was 93.4%.Besides,during the adsorption-desorption recycles,the performance of IRA-900 maintained stable.By applying anion exchange resin in caproate fermentation system,caproate production performance was significantly enhanced.As caproate concentration was removed from fermentation broth,caproate production could be effectively regained and the neat production reached at 16.9 g/L.while the production in the control without adsorption was negligible.Besides,the groups with adsorption could effectively convert butyrate accumulated in the former phase,the neat conversion was 65.8%.The qPCR results revealed that Clostridium kluyveri growth regained from anion resin adsorption was key to caproate production.