Enhanced Strains Breeding And Process Optimization Of Straw Pretreatment Of Biogas Fermentation Based On Genome Rearrangement

With the rapid development of economy and society,the use of primary fuel is increasing.In order to deal with the problem of energy depletion in the future,the development of new clean and renewable energy has become the focus of research.Biogas technology has been applied to industrial production.However,straw pretreatment technology has always restricted the development of straw biogas fermentation industrialization.Therefore,the development of straw pretreatment technology is one of the research focuses of straw resource utilization.Biological pretreatment technology has the advantages of simple operating conditions,no secondary pollution and low cost,which is suitable for the concept of sustainable development in today’s era.In this study,genome rearrangement breeding technology was used to obtain enhanced strains that can efficiently degrade lignocellulose,and the pretreatment conditions were optimized,and the anaerobic biogas fermentation experiment was carried out.The main contents are as follows:1.In order to improve the enzyme production ability of Trichoderma pseudokoningii and Phanerochaete chrysosporium and obtain a variety of genome rearranged parent strains,the original strains were mutated by two mutagenic factors:atmospheric room temperature plasma（ARTP）and diethyl sulfate（DES）.The compound mutagenic conditions of Trichoderma pseudokoningii were determined as ARTP irradiation time of 110 s and DES concentration of 0.8%;The compound mutation conditions of Phanerochaete chrysosporium were ARTP irradiation time of 60 s and DES concentration of 0.6%;Three mutant strains with high enzyme producing ability,TPAD-6,TPAD-55 and TPAD-56,were obtained by rapid screening of mutant strains of Trichoderma pseudokoningii by Congored transparent circle method and determination of cellulase activity;The mutant strains of Phanerochaete chrysosporium were quickly screened by the combination of aniline blue decolorization circle method and guaiacol color circle method,and the lignin enzyme activity was determined.Finally,four mutant strains with high enzyme production ability were obtained,which were PCAD-19,PCAD-26,PCAD-43 and PCAD-83,as the first generation parent strains of genome rearrangement.2.The protoplast preparation and regeneration conditions of Trichoderma pseudokoningii and Phanerochaete chrysosporium were studied respectively.The conditions of protoplast preparation and regeneration of Trichoderma pseudokoningii were determined as follows:the bacterial age was 36 h,the protoplast formation stabilizer was 0.7 mol/L Na Cl,the snail enzyme concentration was 20 mg/m L,and the protoplast regeneration stabilizer was 0.6 mol/L sucrose;The conditions of protoplast preparation and regeneration of Phanerochaete chrysosporium were as follows:the bacterial age was 20 h,the protoplast formed osmotic stabilizer was 0.6 mol/L Mg SO₄,the concentration of wall dissolving enzyme was 10 mg/m L,and the protoplast regenerated osmotic stabilizer was 0.6 mol/L sucrose.The UV inactivation and heat inactivation conditions of protoplasts of Trichoderma pseudokoningii and Phanerochaete chrysosporium were studied respectively.The inactivation conditions of protoplasts of Trichoderma pseudokoningii were determined as follows:UV irradiation for 40 min,50℃water bath for 50 min;The inactivation conditions of protoplasts of Phanerochaete chrysosporium were determined as follows:UV irradiation for 30 min and 50℃water bath for 40 min.The optimum protoplast fusion conditions were determined as follows:the temperature was 35℃,the PEG content was 35%,the fusion time was 10 min and the Ca Cl₂ concentration was 10 mmol/L.Through two rounds of genome rearrangement and genetic stability test,the high-yield cellulase fusion TR2-35 was finally obtained,and its filter paper enzyme activity was 2.45U/m L,which was 226%higher than that of the original strain;the high-yield lignin enzyme fusion PR2-24,The lignin peroxidase activity,manganese peroxidase activity and laccase activity were 11.34 U/m L,88.35 U/m L and 10.82 U/m L respectively,which were 181%,171%and 203%higher than those of the original strain.3.In order to determine the optimal pretreatment conditions of high-yield strains,the optimal pretreatment conditions were determined by orthogonal test:the initial p H value was6.0,the content of corn straw was 2.0%,the inoculation amount of TR2-35 spore solution was 1.0%,and the inoculation amount of PR2-24 spore solution was 0.5%.Under this condition,the weight loss rate of straw after pretreatment for 7 days was 48.67%,which was84.70%higher than that of the original strain.Through the regular detection of the pretreated barber fermentation broth,the optimal pretreatment time was determined,that is on the 8th day after the treatment of high-yield strains,it was most suitable for biogas fermentation.At this time,the p H value of the fermentation broth was 6.8,which was suitable for the growth of anaerobic Methanogens;The COD content in the fermentation broth reaches the highest,which is 10080 mg/L.the straw and its components also reach the peak of degradation,which is most suitable for anaerobic biogas fermentation.By designing a 1L anaerobic fermentation drainage gas gathering device with an effective reaction volume of 800 m L for biogas fermentation,the maximum daily gas production and maximum daily methane content of the high-yield strain pretreatment group were increased by 90.43%and 21.36%respectively compared with the non-pretreatment group,and the maximum daily gas production was 4 days earlier than that of the non-pretreatment group.After 20 days of biogas fermentation,the total gas production and total methane production of the high-yield strain pretreatment group were 6083 m L and 4316 m L,which were 61.14%and 93.63%higher than those of the non-pretreatment group respectively.The results showed that the corn straw pretreated by high-yield strains could effectively improve the biogas yield and shorten the fermentation time,which had a certain economic value.