Bisulfite Pretreatment For Improving Enzymatic Cellulose Digestibility Of Hardwood

Lignocellulose biorefining based on enzymatic cellulose hydrolysis is developed for producing biofuels and biochemicals to substitution of petroleum based products. Chemical pretreatment is the key to effective hydrolysis of lignocellulose substrates and to economic feasibility of the technology. Bisulfite pretreatment has been proved successful in fractionation of most hemicellulose and partial lignin from various lignocellulosic biomass, resulting in significant size reduction and improved cellulose accessibility. However, few study focused on the optimization of process variables with regard to yield of glucose, xylose as well as total monosugar. This thesis was focused on the bisulfite pretreatment technique for promoting substrate accessibility to cellulase for hardwood biomass. The diminishing of cellulase-lignin nonproductive binding was extensively studied in order to further enhance the enzymatic digestibility of bisulfite pretreated lignocellulose.In chapter II, we optimized process variables including chemical dosages, highest temperature and resistance time for bisulfite pretreatment of eucalyptus by using multivariate nonlinear regression. The optimal pretreatment conditions for glucose yield by enzymatic hydrolysis were H₂SO₄ 1.1% (v/v), NaHSO3 4% (w/w), highest temperature 187°C and duration time 15.2 min. The predicted yields of glucose and total monosugar via enzymatic hydrolysis were 100% and 90%, respectively, close to the data obtained under experimental conditions of 180°C and 30 min with the same chemical dosages. However, the optimal conditions for xylose and total monosugar yields differ with that, suggesting the existence of“polydispersity of plant biomass recalcitrance (PPBR)”. PPBR is an inherent feature of lignocellulose biomass which can be determined by simultaneously optimizing yields of cellulose and xylose can hardly be achieved. PPBR can be quantitatively expressed and highly dependent on ratio of xylan to glucan in biomass as well as pretreatment method. The low PPBR suggests low sensitivity of sugar yield to a particular pretreatment variable in bisulfite pretreatment of eucalyptus. The concept of PPBR can help design a two-stage pretreatment especially for those feedstocks with high xylan to glucan ratio.In Chapter III, the optimization was focused on the generation of bioinhibitors, such as lignosulfonates, furfural and hydroxymethylfurfural (HMF). Since the measurement of furfural and HMF by HPLC can be time-consuming, an attenuated total reflection (ATR)-UV spectral method was established for rapidly and simultaneously determining the three bioinhibitors in bisulfite liquor. Surfactant or alkali was used to overcome the interference caused by lignin adsorption on ATR sensor in acidic condition. A single and a dual-wavelength calibration method were used for lignosulfonates quantification. These two calibration methods provide very similar data, which indicates the concentration of HSO₃^- or SO₃^2- in bisulfite liquor was too low to interfere with the ATR-UV detection. The dual-wavelength calibration also provides the total concentration of furfural and HMF, meanwhile a tri-wavelength calibration is necessary for their individual concentration. About 50% of lignosulfonates and 30% of furfural were yielded during the temperature raising period. Significant delignification took place with a low H factor (200-400). To enhance cellulose accessibility to cellulase, delignification seems less important than removal of hemicellulose.In Chapter IV, the inhibitory effects of lignin to enzymatic hydrolysis of cellulose were studied first. The percentage digestibility (PDC) of cellulose was decreased by 15% in the presence of 0.1 g·L^-1 of lignosulfonates because of lignin-cellulase nonproductive binding. The inhibition caused by Kraft or Organosolv lignin was low probably because of their insoluble and hydrophobic characteristics. A critical concentration was found at which over 27% and 11% of PDC was lost due to the presence of lignosulfonates and Organosolv lignin, respectively. Hot water washing can efficiently remove the dissolved lignin that adsorbed on to pretreated biomass. The maximal amount of removable unbound lignosulfonate was almost linearly increased with the washing temperature. The cellulase loading can be reduced by up to 40% as a 98°C hot water washing was applied. The addition of Ca(II) or Mg(II) to a concentration of 10 mmol·L^-1 can also completely eliminate the reduction in PDC caused by the various unbound lignins. The addition of Ca(II) and Mg(II) also reduced the nonproductive binding caused by bound lignin in pretreated wood substrate. The addition of Ca(II) or Mg(II) for unwashed bisulfite pretreated aspen can save as much as 30% of the cellulase loading, equivalent to that by completely washing at 25°C. Applying Ca(II) or Mg(II) significantly eliminates the requirement for washing and therefore, saving considerable process water and cellulase.In Chapter V, an in situ UV-Vis spectrophotometric method was established for rapid and temporally resolved monitoring the adsorption of cellulase on cellulosic and lignocellulosic substrates. A cross-filtering technique was applied to prevent the substrates flow into cuvette. Two spectral analysis techniques were used to correct for spectral interferences from light scattering by fine fibers and from absorptions by lignin leached from lignocelluloses. The wavelength at 280nm was for quantitatively determining cellulase. The dual-wavelength technique uses wavelengths of 500 and 255 nm as secondary wavelengths for cellulose and lignocellulose, respectively. Second derivation of the measured spectra also eliminates the spectral interferences caused by fine fibers and lignin. The kinetic adsorption curve of cellulase onto cellulose and lignocellulose measured by the two in situ spectrophotometric methods showed general agreement with the batch sampling assayed by Bradford method. The measured time-dependent adsorptions of endoglucanase EGV with cellulose binding domains (CBDs) were found to follow pseudo-second-order kinetics. Meanwhile, the adsorption curves of EGI without CBD apparently indicated a significant desorption occurred at an interval of about 7 min. As evidenced by tapping mode atomic force microscopy, it was assumed that CBD enables endoglucanase particles specifically binding to cellulose microfibrils and prevents them aggregate together.