Multiphase Taxonomic Identification Of Lignin-degrading Bacteria And Hydrolysis Of Straw Construction Of Complex Strains

Crop straw is an important biomass resource rich in organic matter(80%～90%).Because of its compact structure,the bioconversion efficiency of microorganisms is affected.Therefore,when using straw as raw material for biogas production,biological pretreatment can destroy the cross-linking degree of lignin,cellulose and hemicellulose in straw,and increase the hydrolysis rate of biogas fermentation to produce acid with low energy consumption and low cost,thus improving methane production.In this study,three strains with high lignin degradation performance were screened by directional isolation using restricted culture technique.The dominant strains were identified by morphological and molecular biological analysis,and the single factor enzyme production conditions of carbon source,nitrogen source and metal ion addition level were optimized for single strain.By comparing the enzyme producing activity,it was determined that the enzyme producing performance of the compound strain was better than that of the single strain.Response surface methodology(RSM)was used to determine the optimal conditions of enzyme production.In addition,compound strains and single strains were applied to the straw hydrolysis test to explore the effects of hydrolysis time and addition amount of bacterial liquid on volatile fatty acid content in hydrolysate and lignocellulose degradation rate in straw.The main results are as follows:(1)Sawed wood samples from Baiguanshan scenic area of Zhejiang Province were selected as samples,and three strains with high lignin degradation ability were obtained through continuous primary screening and rescreening through restricted culture,which were named HJ4,SP8 and LZ10 respectively.HJ4 was identified as Aspergillus terreus by DNA sequence determination of strain 18 S.the laccase activity was(108.01 ± 5.99)U/L,lignin peroxidase activity was(112.04 ± 6.98)U/L.The activity of manganese peroxidase was(97.72 ± 4.55)U/L.The laccase activity of strain SP8 was(106.42±7.43)U/L,lignin peroxidase activity was(89.45±7.21)U/L,manganese peroxidase activity was(113.45±6.99)U/L.The laccase activity,lignin peroxidase activity and manganese peroxidase activity of Fusarium culmorum LZ10 were(95.56±4.56)U/L,(96.33±4.56)U/L and(99.76±5.23)U/L.(2)The optimum carbon source of HJ4,SP8 and LZ10 strains was determined to be soybean straw,and the optimum supplemental amount was 12.5 g/L,10 g/L and 10 g/L,respectively.The best nitrogen source for strain HJ4 and SP8 was soybean meal with 2 g/L and 3g/L,respectively.The best nitrogen source for strain LZ10 was yeast meal with 4 g/L.The optimal potassium supplemental levels of HJ4,SP8 and LZ10 strains were 0.3 g/L,0.2 g/L and 0.3 g/L,respectively.The optimal dosage of copper ion was 0.4g/L,0.3 g/L and 0.2 g/L,and the optimal dosage of iron ion was 0.3 g/L,0.2 g/L and 0.2 g/L,respectively.(3)Response surface methodology(RSM)was used to optimize the enzyme production conditions of the complex strain.The theoretical optimal culture conditions were determined as follows: the effective reaction volume was 78 m L,the inoculation amount was 12.3%,the ratio of carbon to nitrogen was 22.5,and the culture time was 6 days.The laccase activity reached512.76 U/L under the optimal conditions.The reliability of the model can be confirmed by verifying that the experimental results are close to the simulation results.(4)Intermittent experiments were conducted to study the hydrolysis performance of straw by single strain and mixed strain respectively.When the addition amount of bacterial liquid was 8%,the p H value of straw hydrolysate in SP8,HJ4 and LZ10 treatment groups was 4.62,4.61,4.66,and the acetic acid content was 179.36 mmol/L and 185.36 mmol/L,respectively.The p H value of hydrolyzate was 4.21 and acetic acid content was 192.35 mmol/L.The hydrolyzate performance of the mixed strain was better than that of the single strain.