Screening Of Cellulolytic Bacteria And Study On Its Cellulose Degradation Performance In Coconut Oil Cake

Lignocellulosic biomass—the largest renewable energy source—can effectively meet the ever-increasing global energy demands.The cellulose contained in coconut oil cake（COC）,which is a major solid waste of the coconut oil industry,can be converted to alternative energy sources,such as bioethanol,through fermentation.Microorganisms and their carbohydrate-active enzymes are central to the depolymerization of complex lignocellulosic polysaccharides in the global carbon cycle.To develop effective cellulose-degradation systems,microorganisms that can produce appropriate carbohydrate-active enzymes are,therefore,needed to serve as a suitable source of hydrolytic enzyme production for biomass stabilization and bioethanol production.In the present study,we used decaying dahlias as they are a potential low-cost resource pool to screen cellulase high-producing yeasts and bacteria that can effectively utilize COC as the sole carbon source for cellulase secretion through qualitative and quantitative analyses.We also identified strains and explored their enzymatic properties and saccharification efficiency as well as characterized their COC-degradation process to assess the decomposition potential of the isolates.Finally,we evaluated the potential of the screened isolates when co-cultured with Saccharomyces cerevisiae for the production of bioethanol by applying eco-friendly technologies toward resolving the traditional energy shortage.The main results of this study as discussed below:1.A total of 30 strains were isolated and purified from decayed dahlias and screened by qualitative analysis with Congo red and by quantitative analysis with cellulase（CMCase）.We found that the strains MH10 and UE10 demonstrated strong cellulase-hydrolysis ability,with102.96 and 167.3 U/m L of CMCase activity on COC as the sole carbon source.The strain MH10 was tentatively identified as Meyerozyma guillermondii by 18S r DNA and 16S r DNA along with morphological observations.The strain UE10 was identified as Bacillus tropicus.In a liquid medium containing the substrate COC,M.guilliermondii reached a glycation efficiency of 35%at day 20,and the cellulose content in COC was reduced from an initial value of 23.28%to 13.19%.B.tropicus demonstrated a glycation efficiency of 45.42%for the COC substrate,and the cellulose content was reduced to 9.34%.The degradation efficiency indicated the inherent ability of M.guilliermondii and B.tropicus to convert COC into value-added chemicals,such as reducing sugars.2.The effect of temperature,pH,and metal ions(such as Ca²⁺,Fe²⁺,Na⁺,Cu²⁺,Mg²⁺,and K⁺)on CMCase produced by yeast and bacteria was determined by single-factor experiments,and the thermal stability of CMCase produced by the isolates was determined to establish the optimal enzyme reaction conditions.The optimal reaction temperature for cellulase produced by M.guillermondii was detected as 30°C.Among the six metal ions tested,only Ca²⁺showed a slight promoting effect on the cellulase-glycosylation system of M.guillermondii.The thermal stability of CMCase was found to be the highest when stored at 35℃,and the relative enzyme activity was maintained at>85%after 2 h of storage.The relative enzyme activity of B.tropicus was found to peak at 35℃,and the optimum reaction pH was identified as 7.Low concentrations of Mg²⁺,Na⁺,and Ca²⁺demonstrated a promoting effect on the cellulase-glycosylation system of B.tropicus,among which Ca²⁺had the strongest promoting effect,with the relative enzyme activity reaching 118.97%.In addition,Cu²⁺displayed a strong inhibiting effect on the cellulase-glycosylation system.The thermal stability of CMCase was the highest when stored at 40℃,and the relative enzyme activity was found to remain>85.9%after 2 h of storage.3.The COC was characterized before and after degradation through field emission scanning electron microscopy（FESEM）,Fourier-transformed infrared spectroscopy（FTIR）,X-ray diffraction（XRD）,specific surface area and porosity（BET）,thermogravimetry（TG-DTG）,and color difference.FESEM analysis revealed that the surface of COC became rough after biological treatment,with cracked and delaminated substrates and the development of characteristic structures of pores and cracks.Functional group modification by FTIR analysis indicated successful depolymerization of cellulose in COC,such as reduced intensity of the characteristic peaks of lignocellulose at 810.61,1740,2860,and 3400 cm^-1.XRD analysis revealed that the amorphous region of COC was attacked by microorganisms and enzymes,resulting in a significant increase in the Cr I values after the treatment.The thermogravimetric results suggested that the thermal stability and residual carbon rate increased after biological treatment,representing a partial degradation of cellulose and hemicellulose.The color difference analysis confirmed that the L^*and b^*values were much lower than that before degradation and that browning reactions and Maillard reactions occurred during the cellulase hydrolysis by removing some proteins and soluble carbohydrates.Meanwhile,the specific surface area and porosity analysis confirmed that the degraded cellulose became loose and porous,and the BET was enhanced by 162.23-fold and 225.36-fold after saccharification by M.guilliermondii and B.tropicus,respectively.4.B.tropicus was used for the hydrolysis of COC to release reducing sugars.The reducing sugars served as a biofuel resource for the production of bioethanol.The reducing sugar released from the glycosylated COC of B.tropicus reached the maximum yield of 0.9 mg/m L at 48 h,and bioethanol was produced from COC using B.tropicus co-cultured with S.cerevisiae and ethanol.