| Lignocellulosic raw material is an abundant and renewable resource,which is the main source of biofuel and other high value bio-based products.However,hydroxycinnamic acid compounds produced during alkaline pretreatment and subsequent hydrolysis of lignocellulose will seriously affect the bioconversion of lignocellulose,resulting in the decrease of fermentable sugar conversion and ethanol yield.Ferulic acid and p-coumaric acid are the two most toxic hydroxycinnamic acid compounds.In order to explore the mechanism of their inhibition on cellulosic hydrolysis and yeast fermentation,the following studies were made and preliminary results were obtained:1.Effects of FAand p-CA on the hydrolysis of microcrystalline celluloseLow FA can promote cellulose hydrolysis and inhibit hydrolysis when the FA concentration exceeds 0.5 g/L.The hydrolysis of microcrystalline cellulose by p-CA has been inhibited.By infrared spectroscopy and X-ray diffraction analysis,it was found that the addition of FA and p-CA changed the order of hydrogen bond and the crystallinity of cellulose in the compound,and destroyed the secondary structure of protein.SEM showed that the surface of microcrystalline cellulose became uneven and irregularly shaped.The interaction between hydroxycinnamic acid and cellulase was found by ultraviolet spectrum and circular dichroism spectra,which changed the secondary structure of proteins,decreased the content of α helix and increased the content of β fold.The lower concentration of ferulic acid increased the cellulase activity due to the decrease of α helix content,but increased the content of flexible structure-random coil,which increased the affinity of the enzyme to the substrate.2.Inhibitory effects of FA and p-CA on Saccharomyces cerevisiae cellsThe results showed that FA(1.5 g/L)and p-CA(2.0 g/L)could significantly inhibit the growth and glucose utilization of S.cerevisiae.In order to explore the inhibitory effect of FA and p-CA on S.cerevisiae cells,the leakage of electrolyte and intracellular substances was measured respectively.It was found that the integrity of cell membrane was destroyed,permeability was changed,electrolyte,nucleic acid,protein and other contents were released.The cell surface of S.cerevisiae was damaged by SEM,and the cell fold appeared,which affected the normal function of cell wall and cell membrane.Fourier infrared spectroscopy found changes in some molecular groups representing lipids,proteins and nucleic acids,suggesting that FA and the addition of coumaric acid had severely inhibited the normal growth of S.cerevisiae cells.3.The inhibitory mechanism of FAand p-CA on S.cerevisiae was analyzed based on non-targeted metabolomicsThe metabolites of S.cerevisiae cells supplemented with 1.5g/L FAand 2.0g/L pCA will be prepared and extracted,and non-targeted metabolomics analysis will be performed using an performance LC-MS.Based on multivariate statistics such as metabolomics PCA and PLS-DA,100 and 92 different metabolites(VIP value > 1,P value < 0.05)were significantly changed in S.cerevisiae cells treated with FAand p-CA,respectively.Metabolomics analysis showed that the changes of some metabolites with significant differences,including cysteine,L-isoleucine,L-glutamic acid,L-glutamine,L-valine,etc.,disturbed the redox balance in the cells,produced oxidative stress response,destroyed part of the protein synthesis,and was the main reason for inhibiting the growth of yeast cells.Yeast cells,on the other hand,protect themselves from damage by producing more precursor materials and more energy. |
