Bioconversion Of Crude Glycerol To 1,3-propanediol By Anaerobic Microbial Consortium

1,3-Propaneidol is an important bulk biochemical with wide applications on textile,food,pharmacy and cosmetic industries.Biotransfermation of crude glycerol to 1,3-propanediol by microbial fermentation has drawn worldwide attention owing to the high 1,3-propanediol yield,mild reaction conditions and environmentally friendly process.However,natural pure culture always shows low substrate tolerance and limited 1,3-propanediol productivity owing to the inhibitory effect of impurities in crude glycerol and inherent metabolic limitation of natural microorganisms.Alternatively,microbial consortium with inter-and intra-species diversity,is better adapted to complex substrates than pure culture,and the 1,3-propanediol production capacity of microbial consortium is also expected to break through the theoretical yield of pure culture.In this study,an anaerobic microbial consortium C2-2M was selected from anaerobic activated sludge with high adaptation to crude glycerol and overwhelming capacity for 1,3propaendiol production.Using microbial consortium C2-2M as inoculum,the glycerol metabolism,1,3-propanediol production capacity,community structure changes,performance stability as well as potential community interaction were explored in batch,fed-batch and continuous fermentations.The main results were obtained as follows:Firstly,a microbial consortium with effective conversion of crude glycerol to 1,3propanediol was selected from anaerobic activated sludge,and its community structure was identified.The microbial consortium was selected by serial transfer using crude glycerol as the sole carbon source.During the selection process,the microbial consortium underwent metabolic adaptive and fluctuating stages before achieving a stable state based on the growth and metabolism,named C2-2M after 19 serial transfers.Microbial consortium C2-2M showed high adaptation to crude glycerol and competitive capacity for 1,3-propanediol production,with 1,3-propanediol yield of 0.69 mol/mol glycerol,which approached the theoretical yield of pure culture.16S rRNA analysis of microbial consortium C2-2M showed that the family Clostridiaceae represented by C.butyricum occupied the predominant position,accounting for 94.64%.Besides,4.47%Peptostreptococcaceae was found in the consortium.Secondly,the adaptation to crude glycerol,1,3-propanediol production capacity as well as the potential community interaction of microbial consortium C2-2M were explored in batch and fed-batch fermentations.In batch fermentation,microbial consortium C2-2M adapted well with high glycerol concentration to 120 g/L,producing 60.61 g/L 1,3-propanediol with a productivity of 3.79 g/(L·h).In fed-batch fermentation,a large fluctuation of crude glycerol concentration from 15 to 80 g/L had no effect on growth and 1,3-propanediol production of the microbial consortium,with the 1,3-propanediol concentration and productivity of 82.66 g/L and 3.06 g/(L·h),respectively.The 1,3-propanediol production capacity of microbial consortium C2-2M reaches one of the highest levels among natural microorganisms.Furthermore,the community structure of the original microbial consortium was destroyed by serial dilution and single strain isolation to evaluate the potential interaction in the consortium.A series of mini consortia obtained by serial dilution showed similar microbial composition but gradually decreasing tolerance to crude glycerol.Four randomly selected C.butyricum isolated randomly from microbial consortium C2-2M displayed various growth rates and tolerances to crude glycerol,but insufficient 1,3-propanediol production capacities.Based on the above results,it was indicated that the high adaptation to crude glycerol and competitive 1,3-propanediol production of microbial consortium C2-2M was the collaborative effect of different individuals.Thirdly,continuous fermentations under different operating conditions were conducted to evaluate the changing patterns of fermentation performance and community structure of microbial consortium C2-2M.In continuous fermentation at a glycerol feed concentration of 130 g/L and a dilution rate of 0.096 h-1,the highest 1,3-proapnediol production of 57.86 g/L with the productivity of 5.55 g/(L·h)was obtained using microbial consortium C2-2M as inoculum.Analysis of community structures under different operating conditions showed that the community structure became simpler under circumstances of higher dilution rate or higher glycerol feed concentration.Fermentation kinetics showed that microbial consortium maintained a consistent pattern for glycerol consumption and 1,3-propanediol production despite changes in community composition.Furthermore,the performance stability of the microbial consortium C2-2M during long-term continuous fermentation was evaluated.In continuous fermentation under glycerol-excess condition,the microbial consortium was able to operate for a long period of 31 volume changes with an average 1,3-propanediol production of 53.52 g/L.In glycerol-limited continuous fermentation,however,an abnormal oscillatory behavior was observed after continuous operation for over 120 h,along with drastic fluctuation of community structure.Finally,the oscillatory behavior of C.butyricum S3 isolated from microbial consortium C2-2M was comprehensively evaluated in terms of occurrence condition,metabolism and kinetics,redox status,morphology as well as genome and transcriptome.Combined with all existing data,the potential mechanisms of this newly-founded oscillation were proposed.Spontaneous oscillatory behaviors by C.butyricum S3 tended to occur in continuous fermentation under glycerol-limited conditions.C.butyricum S3 exhibited global oscillation among all pathways for glycerol metabolism,in which productions of lactate,formate and H2 were lagged behind that of other metabolites including biomass,1,3-propanediol and butyrate.During oscillation of C.butyricum S3,both intracellular and extracellular redox status exhibited consistent fluctuation patterns,which switched from oxidative to reductive status when cells significant inhibition.Besides,periodic morphological changes were observed during the oscillation,with aggregates and spores or cell debris at the trough of biomass production.Transcriptome analysis indicated that expression levels of multiple genes,including those for pyruvate metabolism,conversion of acetyl-CoA to acetaldehyde as well as stress response,were up-regulated when microbial cells were undergoing stress.Based on the above informations,two hypotheses were put forward to explain this oscillatory behavior:1)disorder of pyruvate metabolism,especially regeneration of ferredoxin;2)excessive accumulation of toxic metabolite,e.g.acetaldehyde.In summary,this study explored the feasibility of using microbial consortium as inoculum for bioconversion of crude glycerol to value-added 1,3-propaneidol,which sets an example for using microbial consortium in other bio-based chemicals fermentation.In the future,rational design from natural to artificial microbial consortium remains to be explored in order to maximize the potential of microbial consortium for industrial application.