Screening Of High Butanol-Producing Strains,Fermentation Process Using Lignocellulose And Analysis Of The Mechanisms Of Phenolic Inhibitor Tolerance

Due to the increasing concern about the issue of global warming and environmental degradation,new interests and greater efforts have been recently devoted to develop the green biofuels,such as bioethanol and biobutanol.Biobutanol is considered the most promising second-generation biofuel as it has many advantages such as high energy content,hydrophobicity and miscibility with gasoline.Therefore,the production of butanol via fermentation has been attracted renew interest in both industrial and academic worlds.However,acetone-butanol-ethanol(ABE)fermentation still faces many problems and challenges such as high feedstock costs and low product concentration or productivity due to the products(especially butanol)toxicity to the producing host.Therefore,ABE fermentation from low costs raw materials and improvement of butanol yield and productivity are the promising strategies enhance its economically competitive processes.In this study,to accelerate the hydrolysis of puerariae slag(PS),the method of ultrasound-assisted dilute acid hydrolysis and acid combined with laccase were development,when PS material was used as the main raw material.Additionally,native strain which could efficiently using PS hydrolysate as the substrate to production butanol was isolated from environmental samples and the yield and productivity of butanol were enhanced by different regulation strategies.Furthermore,the mechanisms of the strain tolerance of the phenolic inhibitors were also analyzed.The main findings of this study are as follows:(1)A total of 75 butanol-producing strains with high butanol tolerance were isolated from 120 soils and sand samples using the“sandwich”screening method,and the strain YBS3 which shows superior fermentation performance was identified as Clostridium beijerinckii by 16S rDNA comparison.A significant phase shift delay was encountered in ABE fermentation process when the PS hydrolysate was used as substrate to produce butanol.To solve the problem of phase shift delay,different C/N ratio regulation strategies were performed.The results show that the problem of the phase shift delay was smoothly solved and the yield and productivity of butanol was improved if the lower C/N ratio was adopted at mid-logarithmic phase.To further improve the fermentation performance of the YBS3,atmospheric and room temperature plasma(ARTP)mutagenesis was used in this study.With this effort,the mutant strain ART44 was obtained,and the productivity of butanol was 0.15g/L/h,which was 25%higher than that of the wild-type strain when the PS hydrolysate was used as substrate.(2)To accelerate the hydrolysis of PS,the method of ultrasound-assisted dilute acid hydrolysis was development.With this effort,the highest hydrolysis efficiency(81.0±0.58%)was obtained under the optimal pretreatment condition.Subsequently,the butanol and total solvent production of 8.79±0.16 g/L and 12.32±0.26 g/L were obtained from the non-detoxified diluted hydrolysate,and the yield and productivity of butanol were 0.19 g/g-reducing sugar and 0.12 g/L/h,respectively.Additionally,the slightly changes in the microscopic surface structure of PS from different pretreatment methods were observed using SEM and FT-IR.The phenomenon of an increase in irregular folds,a decrease in perforation rate of the residues surface and an increase absorption peak during ultrasound-assisted acid hydrolysis process was found,thus resulted in more severe and distinct damage to the dense structure of PS and then enhanced the hydrolysis efficiency.(3)The effect of alone dilute acid(H₂SO₄),alone laccase pretreatment and acid combined with laccase pretreatment on the enzymatic saccharification of PS and ABE fermentation were investigated.The highest reducing sugar concentration of 48.96±1.69g/L was obtained after enzymatic hydrolysis of the PS pretreated by 0.2%H₂SO₄ and 120U/g laccase,which were 12.79%and 65.24%higher than that of the alone acid hydrolysis and alone laccase pretreatment,respectively.Additionally,the production of butanol by simultaneous saccharification co-fermentation(SSCF)and separate hydrolysis and fermentation(SHF)were compared when the hydrolysate of PS with the optimal pretreated condition was used as the substrate.The results show that the productivity of butanol and total solvents via SSCF were reached to 0.15 g/L/h and 0.22 g/L/h,which were 114.29%and120.00%higher than that of the SHF.Furthermore,to preliminary analysis of the pretreatment mechanism,the samples obtained from alone acid pretreatment,alone laccase pretreatment and acid combined with laccase pretreatment were explored by FT-IR,and the results indicate that the method of acid combined with laccase pretreatment could much break the chemical bonds,so it could help cellulase to furtherly approach to the cellulose and then enhance the hydrolysis.(4)The effects of different amino acids on the strain tolerance under the various phenolic inhibitor conditions and the mechanisms of tolerance to phenolic inhibitor stress were investigated.The results show that the production of butanol under butyraldehyde and vanillic acid stress environment was significantly enhanced when aspartic acid and histidine were added at a ratio of 1:3.Under this condition,the production of butanol was 9.67±0.08g/L and 7.35±0.20 g/L,which were 17.21%and 67.05%higher than that of the without amino acids addition groups,respectively.In order to explore the tolerance mechanisms of the strain ART44 with phenolic inhibitors stress,the dynamic change of the cell activity,cell morphology,intracellular NADH and ATP levels,and fatty acid composition of cell membranes were investigated.The results indicate that all phenolic inhibitors could be varying degrees detoxified by the strain ART44.Due to the addition of amino acids,the intracellular NADH and ATP levels were enhanced under the stress environment,and then the biodetoxification of phenol aldehyde or phenolic acid inhibitors could be accelerated.At the same time,the content of saturated fatty acids in the cell membrane was enhanced at the same condition,so the cellular rigidity was improved.This behavior was beneficial to enhance the tolerance capacity of phenolic substances stress.Furthermore,in order to better accommodate the phenolic acids stress environment,the specific surface area of strain ART44 was simultaneously reduced,and so this result could provide an osmotic barrier to improve the tolerance of phenolic acids.