Enrichment Mechanism Of PHA Producing Dominant Bacteria And Optimization Of Three-stage Mixed Culture Process

In addition to the persistent damage to the ecological environment caused by the weak biodegradable nature of petroleum-based plastics,petroleum plastics affect the natural food chain in a new form of pollution such as “plastic particles”,and the impacts of petroleum plastics on the global biosphere become severe and profound over time,thus it is urgent to find scoping strategies.Polyhydroxyalkanoate(PHA)is an intracellular polyester synthesized by bacteria and has physical properties similar to petroleum plastics and biodegradability that petroleum plastics do not possess.At present,mature products in the market mainly come from the pure culture fermentation industry,during the past 20 years,mixture culture(MC)PHA production process based on the environmental biological technology has attracted more attention because of its potential pollutants treatment,resource recovery and low production costs.However,several limiting factors impede the industrialization of MC-PHA production,i.e.,lack of theoretical support for system stability,low synthesis performance,and lack of control strategies for product composition at the overall process level,thus studies were carried out at the levels of microbial ecology,metabolism and process efficiency based on the above limiting factors.Three-stage process is the most mature form of MC-PHA production,and PHA producer enrichment stage is the core of three stage process,the mechanism of this stage is not clear enough and theoretical analysis of its stability is lacking.Temporal variations of MC function and microbial community succession of MCs in the enrichment reactor under F-F mode were firstly monitored,and their relationships were analyzed.An overall similar variation trend of the apparent function were observed after the inoculation in three enrichment reactors fed by different kinds of substrate,i.e.,the MCs demonstrated increasing PHA-accumulating abilities and physical properties changed from transient to steady.An overall similar trajectory of the succession,including the elimination of nonPHA producers and turnover of dominant PHA producers,was also observed.Quantification of pha C showed that,PHA producers could maintain the PHA synthesis of MCs by playing consecutive roles when the highly dynamic turnover occurred.By measuring the variations in EPS composition over time,it was found that the specific physiological characteristics of a PHA producer(genus Thauera)caused fluctuating SVI,but this trait also leaded to its further competitive exclusion by another dominate PHA producer(genus Paracoccus).Selective pressure based on the availability of substrate dominated the entire FF system,resulting in the inevitable microbial community succession in the acclimation phase and maintenance of the stable PHA-accumulating function in the maturation phase.However,predation from bacterial phages most likely led to the unpredictable temporal dynamics of the top competitors.The research confirmed the stability of the F-F enrichment system.The continuous feeding PHA production mode was proposed in this study to overcome the the low substrate utilization efficiency existing in the conventional PHA production stage.The continuous feeding assays under low biomass loading rate(BLR)(in the range of 3.5-5.5 Cmol VFA/Cmol X/d with the corresponding organic loading rate of 3.2-5.3 g COD/g VSS/d)can be directly fed with feedstock with p H from 5.0 to 10.0 without any p H regulation.Higher PHA intracellular content(70.36%)can be obtained owing to that the p H-self regulation state was in the p H range suitable for PHA synthesis.The intracellular PHA content of the MCs was proved to have no correlation with the maximum q PHA,the unique substrate-biomass status(demand exceeded supply)make the substrate efficiently utilized for PHA accumulating(YPHA/S sys of 0.81 Cmol PHA/Cmol VFA).Low biomass output is the cost of steady operation of PHA producer enrichment stage;the resulting low PHA output will keep the product price at high level.An extended cultivation strategy for PHA producing MCs based on the separate feeding of carbon source and nutrients was proposed to solve this contradiction.Under optimized conditions,increased PHA-accumulating ability with a biomass magnification of 43 were obtained.By embedding the extended cultivation process into the production process,theoretically a highly competitive PHA volumetric productivity of 1.21 kg PHA/m3/d can be achieved,which means that to obtain the same biomass yield with the extended cultivation system,the working volume of the selector in the three-stage process needs to increase 53 times.Under the precondition of stable operation and increased production efficiency,the quality of PHA polymer also need to be considered in the large-scale application of MCPHA production process.The ratio of HB and HV monomers in the PHA polymer can significantly affect its physicochemical properties,and the processability can be improved by increasing the relative content of HV monomer.Three kinds of MCs,i.e.,M-Ac,MPr and M-Bu,respectively acclimated using acetic acid type-,propionic acid type-and butyric acid type substrate,were selected.PHA accumulating responses of MCs under varying substrate composition were mainly investigated.Results indicated that,PHA structure was sensitive to the substrate composition,but PHAm of MCs was significantly inhibited when fraction of propionate acid in the substrate increased.Based on the adaptability for the varying substrate composition,three kinds of MCs can be ranked as: M-Pr > M-Ac > M-Bu,and substrate dominated by acetic and propionic acid possessed the optimal regulation of PHA structure.Based on the microbial community structure and metabolic flux analysis,the reason for the different PHA accumulating responses of three kinds of MCs was unraveled,the higher metabolic flux(0.06 Cmol/Cmol X/h)in the pathway where propionyl-Co A translate into acetyl-Co A through decarbonization could explain the the high tolerance of M-Pr to propionic acid in the substrate.It is necessary to find a reliable strategy to regulate the composition of fermented waste carbon liquid under the context of PHA quality optimization.The waste activated sludge was finally used as typical waste carbon to study the strategy of oriented acidification of bio-solid by means of response surface methodology(RSM),and semi-continuous fermentation assays were conducted to verify the reliability and effectiveness of the oriented acidification strategy.