Study On Amino Acids Fermentability And Inhibitor Tolerance Of Corynebacterium Glutamicum Strains Using Lignocellulose Feed Stock

Currently,fermentation medium accounts for large percentage of production cost of biochemicals.Utilization of lignocellulosic material as feedstock for amino acids production could be a proper solution to this difficulty.However,high inhibitors content in the pretreated lignocellulosic material is the major technical bottleneck when lignocellulose is used as feedstock of amino acid fermentation.Inhibitory compounds from pretreatment of lignocellulose feedstock negatively interfere with the consequent enzymatic hydrolysis and fermentation.In this thesis,the difficulty was overcome by several practical approaches on improving amino acids fermentability ofC.glutamicumusing lignocellulosic feedstock.Two C.glutamicum strains were used in this study.C.glutamicum SIIM B460 was used for glutamic acid fermentation and C.glutamicum SIIM B253 was employed for lysine fermentation using freshly pretreated corn stover feedstock without inhibitor removal.L-Glutamic acid is commercially important amino acid that is mainly used as food additive and flavor enhancer in the form of sodium salt.In the first part of study,tolerance and degradation of lignocellulose derived inhibitors were analyzed for C.glutamicum SIIM B460 for glutamic acid production,and strain with enhanced tolerance and degradation capability was evolved by evolutionary adaptation.Inhibitor tolerance of C.glutamicum SIIM B460 for glutamic acid fermentation was analyzed for nine inhibitors in isolation using synthetic media.Strain demonstrated strong tolerance against furans(furfural,5-hydroxymethylfurfural(HMF)),various organic acids(acetic acid,formic acid,levulinic acid),and some of the phenolic compounds(4-hydroxybenzaldehyde,vanillin),but sensitive to coniferyl aldehyde and syringaldehyde.Degradation capability of strain was analyzed for five inhibitors in isolation using synthetic media.C.glutamicum was able to degrade furfural,HMF,vanillin,syringaldehyde,and 4-hydroxybenzaldehyde into the corresponding alcohols and acids with less toxicity to the strain.Evolutionary adaptation approach was developed using inhibitor containing hydrolysate.Adaptation improved the tolerance and degradation capability of the strain and consequently glutamic acid fermentation and cell growth were improved.Finally putative degradation pathways were proposed for furfural,HMF,acetic acid,vanillin,4-hydroxybenzaldehyde and syringaldehyde on the basis of previous studies reported.Lysine is an essential amino acid,is valuable as medicament,chemical agent,food material(food industry),and as feed additive(animal food).In the second part of study tolerance and degradation behavior of C.glutamicumB253 were analyzed and non-biodetoxified corn stover hydrolysate was used for lysine production.C.glutamicum SIIM B253 was employed for lysine production using corn stover hydrolysate.Nine typical inhibitors produced from lignocellulose pretreatment were selected and used for testing the tolerance of strain for lysine fermentation.Strain revealed strong tolerance against nearly all pretreatment inhibitors when used in isolation in synthetic media,including furans(furfural,5-hydroxymethylfurfural(HMF)),organic acids(acetic acid,formic acid,levulinic acid),and phenolic compounds(4-hydroxybenzaldehyde,vanillin,coniferyl aldehyde,syringaldehyde).Tolerance mechanism was investigated by examining the response of C.glutamicum SIIM B253 on biodegradation of furfural,HMF,vanillin,syringaldehyde,and 4-hydroxybenzaldehyde and acetic acid.Lysine fermentation was compared using synthetic media,biodetoxified and non-biodetoxified hydrolysate.In contrast to various other strains,C.glutamicumB253 growth and lysine fermentability was better in terms of tolerance and degradation capacity and produced comparatively higher lysine concentration in non-biodetoxified hydrolysate compared to synthetic medium and biodetoxified hydrolysate.Furfural,HMF,vanillin,4-hydroxybenzaldehyde and acetic acid present in non-biodetoxified hydrolysate were degraded very quickly.Different inoculum ratios did not have any significant difference for lysine production.While revealed strong tolerance of strain to grow and degrade the inhibitors present in non-biodetoxified corn stover hydrolysate even at 1%inoculum ratio.Results revealed that C.glutamicum is potential strain with strong tolerance against pretreatment inhibitors.The research works presented in this thesis show the technical advantages of using C.glutamicum strains for amino acid production using lignocellulose feed stock.Evolutionary adaptation strategy and the inherently tolerant Corynebacterium glutamicum SIIM B460 could be applied for the biorefinery process of inhibitors enriched lignocellulosic material for glutamic acid production.C.glutamicum B253 due to its excellent tolerance and degradation capability,can be used in non-biodetoxified hydrolysate,reducing the biodetoxification step,with high lysine yield saving time and cost.The approach for amino acids production,proposed in this thesis,has benefits at certain range.These advancements certainly provide help on the commercialization of amino acids production from lignocellulosic material.Further efforts are required for development of fast,efficient,and cost effective amino acids production.