Study On The Enhancement Of Caproate Production Via Bioelectrocatalysis And The Mechanism Of Chain Elongation

The over reliance of global production activity on fossil fuels has led to global warming and air pollution.Therefore,to achieve the sustainable development of our society,a renewable resource was urgently needed.Medium-chain fatty acids（MCFAs）were an emerging high-value-added chemical with higher hydrophobicity,lower oxygen-to-carbon ratio,and higher energy density,which could be produced from a variety of low-grade organic waste.In recent years,the synthesis of MCFAs provided a new research orientation for resources/energy exploration from waste biomass.Among them,caproic acid（HCa）is one of the most studied MCFAs.However,current researches for MCFAs were still in its infancy with several bottlenecks like the need for additional electron donors and the limited synthesis efficiency.At present,bio-electrocatalysis（MECs）as a new technology had been applied to the HCa synthesis process,although in a small amount.It can use electrodes to regulate microbial metabolism to enhance the process of anaerobic fermentation.However,the synthetic mechanism and its correlative with the microbe during the electrofermentation process have not been studied in depth.This study determined the optimal electrocatalysis conditions by exploring the HCa synthesis performance and electrochemical characteristics of the electrocatalytic system under different voltages,hydrogen/ethanol reducing power,and different functional bacterial colonization strategies.Then,the diversity and relative abundance of functional microorganisms were analyzed to reveal the microbial mechanism in HCa synthesis.Finally,to explore the performance of MCFAs synthesis and its potential microbial mechanism,a two-stage fermentation system with pre-fermentation-MECs was constructed with waste activated sludge（WAS）as substrates.Firstly,this study explored the performance of HCa synthesis in the MECs system with acetate as a sole substrate under voltages of 0.6 V,0.8 V,1 V,and 1.2 V.The results showed that there was almost no synthesis of HCa at open circuit（OCA）,but with varied amount of HCa synthesis under a closed circuit.At 0.8 V,the HCa production reached a maximum concentration of 500.8±12 mg COD/L with the selectivity of 75.7%.The carbon balance suggested that the carbon loss was less than 10%in the MECs system,with most carbon converting to HCa.The electrons balance further showed that electrons from electrodes account for 9.11%.Furthermore,the maximum current density was generated and the largest double-layer capacitor area was observed at 0.8 V with 2.46 A/m².This indicated that the applied voltage in the MECs system could effectively participate in HCa synthesis.Results of microbial community structure showed that Rhodobacter and Clostridium＿sensu＿stricto accounted for a relatively high abundance.Acetobacterium could synthesize ethanol and participated in the HCa production through the reverseβoxidation（RBO）pathway.Moreover,electrochemically active bacteria（EAB）that could participate in electron transfer was detected,such as Desulfovibrio,Dysgonomonas,and Pseudomonas.Secondly,the effects of the co-existence of ethanol and electrode/hydrogen reducing power on HCa synthesis was investigated,considering the possibility of electron donor/acceptor mixing in actual wastewater or waste biomass.It showed that the maximum HCa yield reached at 7454 mg COD/L with the acetate to ethanol ratio(R_A/E)of 1:3.The corresponding HCa selectivity was 80.9%.The carbon balance and electron balance reached up to 98.6±0.23%and 99.1±7.3%,respectively.Analysis for electrochemical characteristics showed that an oxidation peak at 0.36 V vs.SCE was observed at R_A/E=1:3.Besides,the largest double-layer capacitor area with more capacity of electrons was obtained.Analysis in microbial community structure showed that the anaerobic fermentation bacteria（AFB）reached the highest proportion on the electrode,followed by the homo-acetogen.In addition,Clostridium＿sensu＿stricto and Acetobacterium contributed the most to HCa production,which could consume H₂ and CO₂.Thirdly,to improve the hydrogen reducing power and electron transfer efficiency in HCa synthesis process,the colonization strategy of functional microorganisms in the MECs system was optimized.The mixed flora of electrode active bacteria and HCa synthesizing bacteria was used as electrode colonization flora.The results showed that HCa production in the reactor with mixed bacterial reached the maximum yield（5332.45 mg COD/L）,1.3 times and 8.0 times higher than the reactor with HCa synthesizing functional bacteria and electrode active bacteria,respectively.Besides,the fluctuation in CO₂ concentration in the headspace suggested the a great improvement of hydrogen reducing power during HCa synthesis.Analysis of the electrochemical characteristics showed that the reactor colonized with mixed bacterial reached the highest current value of 1.36 mA,with an obvious redox peak observed compared with the reactors with single bacterial.Further analysis of the electrochemical impedance（EIS）suggested that colonization of mixed bacterial could reduce charge transfer resistance(R_ct)and improve the efficiency of charge transfer.The functional bacteria included AFB,EAB,and Homo-acetogen,which accounted for 31.8%,15.1%,and 5.78%,respectively.The AFB was relatively reduced and the EAB and Homo-acetogen was relatively increased compared with the reactors colonized with only one kind of bacteria.It indicated that the contribution of hydrogen reducing power and the performance of electron transfer in the reactor colonized with mixed bacterial had been significantly improved.Finally,a two-stage fermentation system coupling with pre-fermentation and MECs system was constructed to explore the feasibility and mechanism of organic matter converting and HCa synthesizing.Pre-fermented sludge with different concentrations under the optimized operating parameters was used for substrate.It showed that the experimental groups with a ratio of pre-fermentation/Phosphate buffer solution(R_FWAS/PBS)1:1 obtained the highest HCa selectivity（45.7%）.The utilization of soluble carbohydrate and protein reached to 51.9%and54.7%,respectively.The total release efficiency of carbohydrate and protein in extracellular polymer（EPS）reached to 31.6%and 21.0%,respectively,suggesting the efficient utilization of organic matters in WAS.This result proved that it was feasible to use the MECs system to promote HCa production from pre-fermented WAS.Analysis of electrochemical characteristics showed that R_FWAS/PBS=1:1 reached the peak current of 1.36 m A,with reduced R_ct and the lowest total resistance of 9.99Ω.High-throughput sequencing results showed that Clostridium＿sensu＿stricto and Cetobacterium was relative to HCa producing,with a relative abundance of 10.43%and 16.34%,respectively.The proportions of Macellibacteroides and Petrimonas that could decompose macromolecular organic matter were 5.36%and 4.08%,respectively.In addition,some bacterial related to electron transfer had also been observed.Molecular ecological network analysis also revealed the complex biological relationship between these key bacteria,and the mutual interaction between different bacteria.