Screening Of Microorganism For Effective Utilization Of Straw And The Underlying Mechanism Of Straw Biodegradation

Straw is a rich and inexpensive lignocellulose resource which can be used to produce fuels,chemicals,and other biomaterials.But the presence of lignin in the biopolymeric structure makes it highly resistant to separation and purification of components.At present,the industrial degradation and utilization of straw are still dependent on the processing methods with high pollution and energy consumption,and there is a lack of new technologies that are environmentally friendly,energy-saving,and emission reduction.In the natural environment,some microorganisms have advantages in their potential complex enzymes which can be used for the efficient degradation and conversion of straw.The current work has screened and studied the efficient degradation of straw using targeting microorganisms.Microbial strains with selective delignification activity were used in straw pretreatment to improve the cellulose accessibility to cellulase and bioavailability.Saccharification liquid was prepared with cellulase hydrolysis and it is further used to produces high-value-added microbial lipids.With studies of the straw degradation mechanism in the bio-pretreatment process and the optimization breeding of lignin-degrading strain,a high-value utilization scheme of straw by the microbial origin was established to explore the possibility of application of straw to food and pharmaceutical grade bioindustry.The research work and main results are listed as follows:A large number of potential lignin-degrading microorganisms were screened using guaiacol,aniline blue,and congo red plate oxidizing/decolorizing reactions for potential biodegradation.There were 14 candidate strains obtained by enzyme activity quantitative detection of Lac,Mn P,Li P,CMCase,BGL,and Avicelase in liquid fermentation.Seven basidiomycetes were identified as Pleurotus species.with high ligninolytic activity.Lac and Mn P were expressed simultaneously in PDB culture.P.ostreatus HJ show the highest Lac activity 802.8±6.2 U/L.The SEM characterization showed that the Pleurotus strains were more efficient in the surface degradation of straw,and the erosion of secreted enzymes was the most visible.Seven different bio-materials such as CS,WS,RS were comparatively studied in compositions,physicochemical properties and cultural characteristics.The results showed that the CS,WS and RS contained about 50%of polysaccharide（cellulose and hemicellulose）,low lignin contents（15～19%）,fiber crystallinity is not high and easy to be affected by enzymes,suitable for biodegradation and conversion.Among them,the cultural characteristics of CS and WS are better,and the average growth velocity of cultured mycelia is fast and dense.The white rot degradation caused by P.djamor RP mycelia on CS and WS was the strongest,indicating that part of the lignin structure can be rapidly degraded by RP strain.The screened strains were used for SSF of CS,WS and RS.The biodegradation process of the SSF process was analyzed with lignocellulosic composition,structure,physicochemical properties and enzymatic activity changes.The straw of best lignin degradation was selected for cellulose hydrolysis,and hydrolysis products were analyzed to evaluate the feasibility of white-rot SSF as a pretreatment step.Then it was hydrolyzed with cellulase,the cellulose degradation of RP pretreated CS and C.unicolor SP02 pretreated CS was significantly different.After 20 hours of hydrolysis of cellulase,the glucose release ratio and cellulose hydrolysis rate of RP-CS was increased by 35.6%and 38.0%,respectively,while the hydrolysis of SP02-CS was lower than untreated CS.The characteristics of enzyme activity and material changes showed that RP and SP02 had different biodegradation mechanisms of straw.In CS,the Lac express of RP was earlier and entered the secretory peak on the 6^th day,while the Li P was secreted later and reached the secretory peak on the12^th day.The synergic effect of ligninolytic enzymes led to a great change in the structure of RP-CS during fermentation period of 18^th to 30^th day,and the cellulose was rarely degraded in this stage.And the straw material has more oligomeric,reunited,amorphous cellulose and other easy-to-hydrolysis polysaccharide structure.Therefore,it can be considered that the degradation of straw by RP is a desirable delignification strategy,which is suitable for the biological pretreatment of straw.SP02 showed effective simultaneous degradation of components.Genetic analysis showed that SP02 was rich in carbohydrate-degrading enzymes,which led to excessive consumption of oligomer structure and affected the subsequent enzymatic hydrolysis and fermentation utilization.In order to improve the lignin-degradation efficiency of straw-pretreating strain,the protoplast preparation and ARTP mutagenesis conditions of RP were studied to construct a directional mutagenesis breeding system.The results showed that ARTP mutagenesis method had better biological effects on protoplasts prepared with RP mycelium.The mutagenesis breeding system is suitable for RP breeding optimization.The RP protoplasts were mutated and screened by the mutagenesis system,and the Lac activity,growth rate and lignin degradation efficiency of the screened mutants were enhanced compared with the wild strain RP.Under the SSF condition,the Lac activity of mutant strains 62-27 was the highest,which was 30.28%higher than that of RP increased by 30.28%.Lac activity improvement enhanced lignin degradation efficiency,but the hydrolyzability of the pretreated straw did not change significantly compared with the wild strain RP.RP-CS was hydrolyzed with cellulase to prepare liquid carbon source for secondary fermentation of oleaginous microorganisms.On this basis,a step fermentation system was established to convert straw into microbial lipids.The results showed that the carbon source of RP-CS hydrolysate had a good effect on the growth and lipid accumulation of oleaginous microorganism Aurantiochytrium sp.SW1,and can be used as a glucose substitute carbon source for the lipid fermentation of SW1.The biomass of SW1 was increased by 62%,and the yield of DHA was increased by 10.5%with RP-CS enzymatic hydrolysate carbon source.In this research,microbial screening,straw pretreatment,mutagenic breeding,and step fermentation were studied to build a biological route of the straw bioconversion system to produce microbial lipids.The research provides a theoretical basis and technical support for expanding the application range of straw resource and also lays a preliminary foundation for building a microbial cell factory for consolidated bioprocessing of straw.